VITAMINS
`
`241
`
`oceurs in two forms, dextrorotatory and levorotatory, and of these only
`the dextrorotatory form has biological activity.
`Since free pantothenic
`acid can readily be obtained only as a sirupy, gummymass, it is usually
`converted to the ealeium salt, which is a white powder and the form in
`which the synthetic product is supplied.
`Since it is an amide, pantothenie acid is readily hydrolyzed by heating
`in either acid or alkaline solution. Hydrolysis results in complete de-
`struction of the vitaminactivity.
`It is rather stable to boiling in neutral
`aqueous solutions, althoughit is destroyed by long heating at 120°C,
`It appears that pantothenic acid is not extensively destroyed by ordi-
`nary cooking of food. Losses of approximately 50 per cent may, however,
`oceur if the cooking water from vegetables is discarded.
`
`Occurrence
`
`liver, ege yolk, and rice polishings are very rich sources of
`Yeast,
`pantothenie acid, while dairy products, whole cereals, muscle meats,
`green leafy vegetables, and certain other vegetables like cauliflower and
`sweet potato, may be classed as good sources. Fruits and egg white are
`low in pantothenie acid.
`The assay of foods for this vitamin is based on the growth response
`of chicks when fed the test material. A bacterial method very similar
`to the one described above for riboflavin has also been developed. The
`human requirement for pantothenic acid has not yet been determined,
`but it has been suggested that about 10 mg. per dayis adequate.
`
`PYRIDOXINE (VITAMIN B,)
`
`Physiological function
`
`Rats receiving an inadequate supply of this vitamin develop a derma-
`titis, which makes its appearance in a characteristic manner. The paws
`and tips of the ears and noseare first affected, becoming red and swollen.
`The area immediately surrounding the nostrils becomes bare, and there
`may be a nasal discharge. The administration of pure pyridoxine ma-
`terially improves the condition of the rat, but even more striking improve-
`ment results from the use of certain fats, especially those which supply
`the so-called “essential fatty acids.” The relation between the physio-
`logical action of these fatty acids and pyridoxine is not yet clear.
`It
`may well be that both are required for the normal nutrition of the rat.
`Neither black-tongue, pellagra, nor chick dermatitis is cured by pyri-
`doxine.
`It has been shown, however,
`that pyridoxine is required by
`dogs, swine, pigeons, and chickens, and several reports indicate that it
`is also important in human nutrition. Deficiency symptoms that have
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 245
`Petitioner Microsoft Corporation - Ex. 1032, p. 245
`
`

`

`242
`
`VITAMINS
`
`been encountered in various animals include a type of anemia andfits
`resembling epileptic seizures in human beings. Pyridoxinedeficiency in
`man has been observed in a numberof cases of pellagrins whostill were
`not completely well after reeciving nicotinie acid, thiamineand riboflavin,
`Symptoms noted in such patients were nervousness, irritability, abdominal
`pain, weakness, and difficulty in walking. These symptoms were quickly
`relieved by the use of synthetic pyridoxine,
`
`Chemical nature
`
`Pyridoxine, or vitamin Bg, was first isolated as a pure chemical sub-
`
`Saar a ee
`
`stance in 1938, and during the next year it was prepared synthetically. ree
`
`Courtesy of Merck & Co.,
`
`Ine,
`
`Fig. 9-12. Pyridoxine.
`
`The chemical nature of this vitamin is best expressed by its structural
`formula:
`
`co
`C
`—c74Sc—cH,
`HO—C7,,)
`§C—CH.OH
`(2)
`(6
`CH—CL0H
`
`Pyridoxine, CsH;,NO3
`Note that it is related to nicotinic acid in that it is a pyridine derivative.
`The name pyridoxineis derived from the chemical namefor this substance,
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 246
`Petitioner Microsoft Corporation - Ex. 1032, p. 246
`
`

`

`VITAMINS
`
`243
`
`which is 2-methyl-3-hydroxy-4,5-di- (hydroxy metlivl)-pyridine.
`Two closely related substances, pyridoxal and pyridoxamine, are repre-
`sented by the following formulas:
`co
`HO—C~°Sc—CH.OH
`HCC.CH
`
`oes
`HO—C~°Sc—cH.OH
`H.-C,20H
`
`Pyridoxal, C;H,0;N
`Pyridoxamine, C;H,,0.N,
`These substanees have about the same vitamin Bg activity for animals and
`for yeast cells as pyridoxine does, but are several thousand times more
`effeetive for certain baeteria. A phosphorylated derivative, pyridoxal
`phosphate, functions as a coenzyme for enzyme systems present in many
`bacteria, whieh break down amino acids into the corresponding amines
`by removing carbon dioxide from the carboxyl group of the amino acid
`(p. 321).
`It is therefore called a codecarboxylase. Both pyridoxal phos-
`phate and pyridoxamine phosphate function as coenzymesin certain trans-
`amination reactions (p. 343) and may, therefore, be called cotransami-
`nases.
`
`CHO
`
`HO—C7 ~C—CH,0—PO;H,
`H,C—Cx,CH
`Pyridoxal phosphate
`
`CH.NH,
`
`é
`HO—G# “GCEFOHs
`H,C—CxyCH
`Pyridoxamine phosphate
`
`Pyridoxal phosphate also serves as a coenzyme for the enzyme system
`involved in the synthesis of tryptophan by a certain mold species (Neuro-
`spora crassa).
`It is,
`therefore, quite clear that the Bg vitamins play
`important roles in both the decomposition, interconversion, and synthesis
`of aminoacids in living cells.
`Pyridoxine is stable to heat, alkalies, and strong acids, but is rather
`easily attacked by oxidizing agents. As yet little work has been done
`on its destruetion during the cooking of food. No reliable figure for the
`human requirement is available, but a tentative value of 1.5 mg. per
`day las been suggested.
`
`Occurrence
`
`Pyridoxine is present in yeast, bran and embryoof cereal grains, meats,
`milk, and leafy vegetables. The amount of this vitamin in a number
`of common foods is given in Table 9-5.
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 247
`Petitioner Microsoft Corporation - Ex. 1032, p. 247
`
`

`

`244.
`
`VITAMINS
`
`Table 9-5
`
`Pyridoxine content of common foods
`
`Beef, lean 2.2... cece cece cece suuecusensseucveuwnes
`Beek Uivers Gaansiaacs
`se
`ere ate ee ers eee Ee ea ge
`Bread, white 2.020.000.0000 ccc cece vee ecceceeccceuvevvveeseves
`Bread;:whole wheat.
`252acc8eesieiigesss HemecnGuxs sc eoneremnes
`Cabbage sian asamnace
`ssisee
`eis eacasteancas ai eeleatr eae eer :
`CaO erento eter erect nnr er evarere rete a nea eno een
`Chicken; datk: meat sscccescascieess ereacere Gn ree eee
`Lamb, leg of . 20.0... ccc cece ccc cece eee cere en ew ee renee enaes
`Milk, whole
`|... 2.0000 e eee eect ee eeentaes
`Onfities)) caccsssianeniceneaaanss ete teasing nanees ae neon
`Pork loin 2.0.0.0... eee eee eee ee TE ey ee
`Potatoes white sacteacss esas eee Reece Eee Cee ae
`Veast, Gnied brewer's success eiecceecaeuiscpntacteateea rec etacaimenees
`
`Milligrams per 100 g.
`edible portion
`0.40
`0.73
`0.30
`0.70
`0.29
`0.19
`0.20
`0.38
`0.20
`0.25
`0.60
`0.16
`5.5
`
`BIOTIN
`
`This member of the vitamin B complex is a substance which has been
`variously knownas “coenzyme R,” ‘“‘vitamin H,” “biotin,” and the “anti-
`egg white injury factor.’
`It was first obtained in pure form and given
`the name biotin in 1986 by Koégl, who was studying it as one of the
`vitamin-like substances required for normal yeast growth.
`
`Physiological function
`
`The feeding of biotin brings about the cure of a nutritional disease
`which develops when rats, chickens, or human beings consume large
`amounts of raw egg white. This “egg white injury” disease is primarily
`a dermatitis, characterized in the rat by swelling and inflammation of
`the skin, especially around the mouth, and by loss of hair. The disease
`is actually an induced biotin deficiency caused by the combination of
`the biotin normally present in the food with a particular protein, avidin,
`present in raw egg white. When so combined, biotin cannot be absorbed
`and utilized by the animal organism. Cooked egg white on the other
`handis perfeetly safe in the diet, since heating to 100°C. destroys the
`ability of avidin to combine with the vitamin.
`Although the above facts demonstrate that biotin is an indispensable
`nutrient, it has not been possible to produee the “egg white injury” dis-
`case in rats by feeding them diets extremely low in biotin. Apparently
`a sufficient supply of the vitamin to meet
`the needs of the animal is
`synthesized by bacteria in the intestinal tract. However, this deficiency
`can be produced in the chick without the use of raw egg white. Like-
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 248
`Petitioner Microsoft Corporation - Ex. 1032, p. 248
`
`

`

`VITAMINS
`
`245
`
`wise many of the lower organisms such as yeasts, bacteria, and fungi
`do require biotin for normal development. No biotin deficieney has been
`observed in human beings consuming their customary dicts. The daily
`intake of biotin on an average diet ranges from 25 to 50 pg., and the
`urine and feces together may contain from two to five times these
`
`quantities.
`
`Courtesy of the SM. A, Corporation,
`
`Fig. 9-13. Biotin,
`
`Biotin appears to function (possibly in the form of a coenzyme,al-
`though none has yet been identified) as a catalyst for cne of the reac-
`tions of the citric acid cycle (p. 330):
`
`COz + CH;,—CO—COOH —> HOOC—CH.—CO—COOH
`Pyruvie acid
`Oxalacetie acid
`
`Further, certain lactobacilli which normally require biotin grow well with-
`out it if oleie acid is supplied instead. This observation indicates some
`kind of a metabolic relationship between these two substances, perhaps
`participation of biotin in the biosynthesis of oleic acid. The vitamin is
`also required for deamination by bacterial cells of serine, threonine, and
`aspartic acid.
`;
`
`Chemical nature
`
`Biotin has a two ring structure with a side chain attached to one of
`the rings.
`It is an acid, as is indicated by the carboxyl group in the
`side chain. Note the urea-like structurein one of the rings (A) and the
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 249
`Petitioner Microsoft Corporation - Ex. 1032, p. 249
`
`
`
`

`

`246
`
`VITAMINS
`
`presence of sulfur in the other ring (6). Biotin and thiamine are the only
`vitamins that contain sulfur.
`
`CH—(CH 2) i— COOH
`
`Biotin, CijH,,0O;N.5
`
`Although it is readily destroyed by such oxidizing agents as bydrogen
`peroxide, biotin is, in general, a very stable substance.
`It is not affected
`by light, strong acids such as normal HCI or HzSO,, nor by exposure to a
`degree of heat greater than that encountered during ordinary cooking
`operations. However, it is destroyed by strong alkali. In manytissues
`it appears notto exist in a free state, but in combination with some cell
`constituent, presumably protein. This view is supported by the recent
`isolation from autolyzing yeast of biocytin, a peptide-like combination
`of biotin and the aminoacid, lysine. Note that the linkage is through
`the epsilon amino group of lysine.
`
`tC
`
`.
`HN~ NH
`|
`HC———CH
`
`asetCDCONE(Heeacon
`
`NH,
`
`Biocytin
`
`Nothing is known as yet regarding the amount of biotin needed by
`human beings. However, the quantities required by various lower organ-
`isms are so extremely minute that it must be regarded as one of the
`most highly active substances known.
`Its effect on yeast growth, for
`example, can still be detected at dilutions of 1:300,000,000,000.
`
`PTEROYLGLUTAMIC ACID
`
`This vitamin was first observed in connection with studies on the nutri-
`tional requirementsof lactic acid bacteria. An impure preparation from
`liver, designated as the “norite eluate factor,” was shown to be necessary,
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 250
`Petitioner Microsoft Corporation - Ex. 1032, p. 250
`
`

`

`VITAMINS
`
`247
`
`in addition to previously known vitamins, for the normal growth of these
`organisms. The effective substance present
`in such preparations was
`later found to be identical with “factor U" and “vitamin M,” which at
`that time were still unidentified, but were recognized as dietary essentials
`for chicks and monkeys, respectively. Other investigators, working with
`various experimental animals, proposed still other names for vitamin-
`like substances which eventually turned out to be pteroylglutamie acid,
`or closely related compounds. These names included “vitamin B,,”
`“factor R,” “factor S,” “folie acid,” “S. lactis R factor or SLR factor,”
`“liver L. casezt factor.” The term folie acid is still in use, but should
`nowbe replaced by the proper chemical names (see below).
`
`Chemical nature
`
`Pteroylglutamie acid is a complex substance made upof three parts,
`glutamic acid, para-aminobenzoic acid (p. 254), and a pterin, chemically
`linked together:
`
`HC
`
`H
`
`Ha—Egee a—CONHCHCH,CH,COOH
`NXO920-CHNH—C.NeH
`COOH
`
`yt
`
`l
`OH
`
`Pteroylglutamie acid
`
`This substance is identical with the “liver L. casei factor,” vitamin B.,
`and folie acid. The name folacin was proposed in 1949 by the American
`Institute of Nutrition as a synonym for folic acid. The “fermentation
`L. casei factor” is very similarly constituted except that three glutamic
`acid residues are present.
`In this form, also called teropterin, the second
`and third glutamic acid residues are linked to the preceding one through
`the gamma carboxyl group rather than through the alpha carboxyl
`(see
`p. 1381). This is the same type of peptide linkage as is found in gluta-
`thione. Still another form, vitamin B, conjugate, contains seven glutamic
`acid residues. The SLR factor, or rhizopterin, contains no glutamic acid
`at all but bears an aldehyde or formyl group on the nitrogen atom in
`position 10:
`
`N
`
`NHeorage oe.
`i *£
`NQwC.@Z0—CH-N-G_
`E—COOH
`|
`=CH
`OH
`Formy|pteroic acid (or rhizopterin)
`
`6)
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 251
`Petitioner Microsoft Corporation - Ex. 1032, p. 251
`
`

`

`246
`
`VITAMINS
`
`The glutamie acid derivative of rhizopterin, formylpteroylglutamie acid,
`or formyl] folic acid, has been prepared synthetically and found to possess
`the typical vitamin activity of other members of this group.
`It probably
`also occurs naturally. Rhizopterin itself, however, does not relieve the
`symptomsof folic acid deficiency in higher animals.
`Very recently a substance needed for normal growth of the bacterium
`Leuconostoc citrovorum (the so-called “citrovorum factor’) has been
`found to be closely related to formylpteroylglutamic acid, from which
`it can be obtained by reducing and heating. The product, namedfolinic
`acid by one group of investigators and leucovorin by another, has been
`shown (Consulich, et al., Pohland, et al.) to have the following formula:
`
`N
`
`H
`N
`
`Ho
`
`coon
`o-G
`H.N—C7 “ee hi
`Ny CX YCCHING
`(C—CONHCHCH.CH.COOH
`¢
` Y
`c=¢
`OH
`CHO
`H
`H
`Folinic acid (or leucovorin)
`In manytests folinie acid possesses higher activity than other members
`of the folic acid group.
`It may be the metabolically active (coenzyme)
`form of this vitamin, or at least it may be more closely related to the
`coenzyme than pteroylglutamie acid itself.
`
`Physiological function
`
`This vitamin is essential for a wide variety of living organisms, and,
`in fact, is probably needed byall living cells. The outstanding deficiency
`symptoms in higher forms (mammals, birds) are anemia, leucopenia (a
`reduced number of white blood cells), weight loss, oral lesions, and diar-
`rhea.
`In the chick the deficiency also results in abnormally poor
`feathering.
`That several hunian diseases are the result of a lack of pteroylglutamic
`acid orrelated substances is indicated by the improvement which follows
`their administration. The best example is sprue, a disease characterized
`by macrocytic anemia (enlarged red blood cells),
`leucopenia, glossitis
`(inflammation of the tongue), diarrhea with large amounts of fatty ma-
`terial in the feces, weight loss, and poor absorption of food from the intes-
`tine. Daily doses of 10 mg.of pteroylglutamicacid orof the triglutamate,
`teropterin, result in prompt relief of these symptoms. Related conditions
`described as nutritional macrocytic anemia and macroeytic anemia of
`pregnancy are similarly benefited. Pernicious anemia patients are bene-
`fited somewhat, but the improvement is temporary and incomplete,
`in
`contrast to the effects of vitamin Bz (see below).
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 252
`Petitioner Microsoft Corporation - Ex. 1032, p. 252
`
`

`

`VITAMINS
`
`249
`
`In all of the above diseases the administration of relatively large daily
`doses (about 4 ¢g.) of a simple pyrimidine compound, namely thymine
`(p. 155), has an almost equally beneficial result. From this and other
`evidence it seems probable that the biological function of pteroylglutamic
`acid is concerned with the biosynthesis of thymine and other components
`of nucleic acids. Teropterin has been claimed to relieve pain in advanced
`eases of human eancerandto retard the growthof tumors in experimental
`animals...
`io
`
`Food sources and requirements
`
`a
`a?
`
`The pteroylglutamic acids are rather sensitive substances which may
`be quite largely destroyed during the cooking of foods. Losses of 50
`to 90 per cent have been reported in meats cooked in different ways.
`Vegetables kept for three days at room temperature lost 20 to 80 per
`cent, and large losses occurred during canning. When asolution of the
`pure vitamin was placed in bright daylight for 8 hours, 88 per cent was
`destroyed.
`a
`é
`According to Toepfer and co-workers a number of common foods may
`be trouped as follows, on-the basis of the milligrams of folie acid which
`they contain per 100 g. of dry weight: Over1.0,5brewer’s yeast, chicken
`liver_psparagus, broadleaf endive, broccoli, leadTetiuce, spinach; 0.4-1.0,
`most' of the other leafy greens,
`liver, blackeye peas, dried beans, soy
`flour; 0.10.4, other vegetables except root vegetables and a fewfruits;
`0.03-0.1, root vegetables, most fresh fruits, grains and grain products,
`nuts, lean beef; 0.03 or less, eggs, milk, meats (other than beef), poultry.
`The amount of pteroylglutamic acid normally required by human
`beings has not been established. Various animal species need 0.005 to
`0.06 mg. per kilogram of body weight per day.
`
`VITAMIN By,»
`
`It has long been recognized that liver and suitable extracts prepared
`from liver contain some substance whichis effective in the treatment
`of pernicious anemia, a serious, wasting disease of man, which if untreated
`is invariably fatal. Many efforts to isolate and identify the “antiper-
`nicious anemia factor” in liver have been made. With the discovery
`of pteroylglutamic acid and the observation that it is effective in curing
`certain pathological blood conditions,
`it seemed that
`the long-sought
`substance might have been found. However, continued treatment of per-
`nicious anemia patients with pteroylglutamie acid proved disappointing,
`since the initial improvement did not last and was often followed by
`severe neurological complications.
`Finally, in 1948, a red crystalline substance was isolated from liver
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 253
`Petitioner Microsoft Corporation - Ex. 1032, p. 253
`
`

`

`250
`
`VITAMINS
`
`which proved to be effective against pernicious anemia in amazingly
`small doses
`(Fig. 9-14). The new material, designated vitamin Bj»,
`contained4.4 per cent cobalt, 2.3 per cent phosphorus and had the formula
`
`Cei-6sHs6-92N14013PCo (inolecular weight about 1350). Although the =
`
`¢
`
`Sy
`
`v
`
`*
`
`*
`
`
`
`Se
`
`>. ee.
`
`zi
`
`= Me tn
`
`a
`-
`:
`Courtesy of Abbott Laboratories.
`
`Fig. 9-14. Vitamin By erystals (x 200).
`
`complete structure is not yet known, several fragments of the molecule,
`including 5,6-dimethyl benzimidazole, have been identified after acid
`hydrolysis. Surprisingly, this substance itself showed full vitamin By2
`activity for rat growth when tested in 5 mg. daily doses. Other hydrolysis
`
`HogSeo
`Hobs, sTe 5a
`
`H
`
`H
`
`5,6-Dimethy] benzimidazole
`
`products identified are propanolamine (CHsCHOHCH2NH¢»)and a phos-
`phorylated derivative of the 5,6-dimethylbenzimidazole (ribose-3-phos-
`phate attached to the N at position 1). Vitamin Bie also contains a
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 254
`Petitioner Microsoft Corporation - Ex. 1032, p. 254
`
`

`

`VITAMINS
`
`251
`
`eyanide group (CN) bound in a coordination complex with the cobalt
`atom, which can be replaced by Cl, 804, OH, SCN, or other groups to
`produce analogs of the natural substanee. The analog containing the
`water molecule has been called vitamin Bis, and is apparently identical
`with another preparation provisionaliy designated By». Brink and co-
`workers have suggested that the Biz molecule, except
`for the eyanide
`group, be called cobalamin. By this nomenclature, vitamin By» would
`be named cyano-cobalamin and Biza, hydroxo-cobalamin. All of these
`various forms of the vitamin have approximately the same kind and
`amount of biological activity.
`
`Physiological function
`
`In the short period since its isolation vitamin By, has acquired excep-
`tional practical
`importanee because of
`its demonstrated usefulness in
`pernicious anemia and related diseases, in livestock feeding, and in human
`nutrition.
`Its absence from the tissues of the body is apparently the
`specific cause of pernicious anemia.
`Injection of as little as 1 pg. per
`day dramatically alleviates the symptoms of this disease.
`It
`is less
`effective when given by mouth because pernicious anemia patients lack
`some substance (“intrinsie factor”)
`in the gastric juice which protects
`vitamin Bye and favors its absorption. Small doses of vitamin Bye are
`also effective in sprue and other macrocytie anemias.
`See Plate IV op-
`posite p. 223.
`It has been known for many years that animal protein supplements
`(e.g., meat seraps, dried whey,ete.) used in livestock feeding contain some
`factor necessary for growth of animals fed only plant proteins. This un-
`known substance was called the animal protein factor (APF). Vitamin
`Bye is certainly the chief and, perhaps, the only component of APF.
`Because of its high APF potency, it is now widely used in animal feeds.
`Availability of vitamin Bys has madepossible the use of larger proportions
`of the relatively cheap plant protein concentrates
`(soybean,
`linseed,
`cottonseed meals), which are more plentiful
`than those from animal
`sources, and has thus been a boon to livestock production.
`The vitamin Biz used in feeds is obtained almost exclusively from
`fermentation sources, and especially as a by-product’ of the fermentations
`which produce such antibiotics as aureomycin, terramycin, and strepto-
`mycin.
`It was noted that crude Biz concentrates from these sources gave
`greater growth responses in some species than could be accounted for by
`their By. content. The extra effect was traced to the antibioties still
`present as impurities in the eoncentrates. This discovery has opened
`newvistas in the science of nutrition, since by use of this combination
`faster growth rates have been achieved than had previously been con-
`sidered optimal on the best mixtures of natural
`foods. The effect
`is
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 255
`Petitioner Microsoft Corporation - Ex. 1032, p. 255
`
`

`

`252
`
`VITAMINS
`
`shown by a wide variety of antibacterial agents and is probably due to
`destruction of intestinal microorganisms which otherwise compete with
`the animal for essential food factors.
`Very recently Wetzel et al. have reported that doses of 10 wg. of vita-
`min Byes given daily by mouth to a group of malnourished school children
`resulted in definite stimulation of growth in 5 of the 11 eases treated.
`These results establish the existence of human vitamin By deficiency
`other than that of pernicious anemia. Howextensive this may be remains
`to be determined by further study, but present indications are that vita-
`min By. may well prove to have wide applications in human nutrition.
`The metabolie function of vitamin By2 in the animal bodyis evidently
`closely related to that of pteroylglutamie acid (for example, both are
`effective in certain types of anemia). Specifically, vitamin By. appears
`to take part in the biosynthesis of nucleic acids and in the formation
`and use of active methyl groups in the body (for example, in the forma-
`tion of methionine from homocystine).
`
`Food sources and requirements
`
`As already indicated, vitamin Biz is more concentrated in foods of
`animal origin than in plant products, and relatively large amounts are
`formed during the growth of many microorganisms. The distribution of
`this vitamin in various foods, as determined by Elvehjem and co-workers
`by means of a rat assay method, is shown in Table 9-6. No figure for
`the normal human requirement for vitamin By» has been established, but
`1 pg. per day,
`if injected,
`is sufficient
`to maintain pernicious anemia
`patients in good condition. This amount is muchless than the minimum
`human requirement, of any other vitamin or trace element.
`Table 9-6
`
`Vitamin By content of foods
`
`(Micrograms per 100 g., fresh basis)
`Minimum vitamin
`Biz content
`*
`*
`
`Foon
`Fore VOU sca enae are
`Gioavisemuk: science
`Green peas ...... hres
`Horse meat, canned .
`.
`Mutton ............
`Pork, shoulder .......
`Bork Dain 22e sete see
`Potatoes ss...
`Tomato juice ........
`VER ose oesmsoreeecasecorerereys
`
`Minimum vitamin
`Bi content
`1.4
`*
`>
`34
`3
`11-2
`12
`*
`*
`2
`
`Foop
`Barley ..............
`TRGANSP aes ae cieterciers
`7
`Beef, liver ...........
`Beef, kidney .........
`Beef, round, cooked.
`Beef,tongue .........
`Cabbage <.sdienou.
`Cheddarcheese ...
`Chicken liver .......
`Cow'simuilk-- asses
`* No measureable amount.
`
`15
`20
`2-3
`3
`*
`14
`11
`Trace
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 256
`Petitioner Microsoft Corporation - Ex. 1032, p. 256
`
`

`

`VITAMINS
`
`CHOLINE
`
`253
`
`Physiological function
`
`A lack of choline in the dict of young, rapidly growing rats results in
`the accumulation of excessive amounts of fat in the liver. There may
`also be damage to the kidneys, which become discolored from internal
`hemorrhage. The ‘fatty livers” are restored to normal by feeding small
`amounts of choline or of methionine. On the other hand, feeding choles-
`terol aggravates the condition. Older rats are muchless likely to suffer
`from the symptoms of choline deficiency.
`It is supposed that the fatty deposits in the liver are caused partly
`by a failure of fat transport and partly by a decrease in the normal
`ate of fat catabolism (that is, transformation into other simpler ma-
`terials) in the liver. The evidence at present available is consistent with
`the assumption that neutral fat (that is, glycerides) must be converted
`into phospholipides before it is transported elsewhere in the body or, if
`it remains in the liver, before it
`is catabolized. Since choline is one
`component of the lecithin type of phospholipides, it would obviously be
`needed for these purposes.
`In faet, it has been possible with the aid of
`radioactive phosphorus to follow the rate of “phospholipide turnover”
`in the liver, that is, the rate at which phospholipide molecules are formed
`and removed, and to demonstrate that choline increases this rate. The
`effect was observed within one hour and was proportional to the amount
`of choline fed.
`Choline is also required for the normal nutrition of chicks and of
`young turkeys.
`In conjunction with manganese it prevents the develop-
`ment of a disease of chickens known as perosis, in which the leg tendon
`slips off from the hock joint as a result of malformation of the bone, and
`the bird is consequently unable to walk. Normal egg production by
`chickens is also impaired by a lack of sufficient choline in the diet.
`One of the main metabolic functions of choline is to supply “labile”
`methyl groups for various transmethylation reactions. These are de-
`seribed in Chap, 18.
`
`Chemical nature
`
`Choline is a very strong basc, with the following suructural formula:
`
`yes
`HOCH,—CH,—Nats
`HO CH;
`
`Choline
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 257
`Petitioner Microsoft Corporation - Ex. 1032, p. 257
`
`

`

`254
`
`VITAMINS
`
`Like nicotinic acid it had been known to organic chemists and had been
`obtained synthetically long before its usefulness as a vitamin was dis-
`covered.
`It is very soluble in water and is quite stable to boiling in
`dilute aqueous solution. Hot alkalies, however, decompose it with the
`formation of trimethylamine.
`Bound choline in the form of lecithin is present in every living cell,
`and free choline is likewise very widelydistributed in biological materials.
`At present, no information is available regarding the human requirement
`for this dietary factor.
`
`OTHER DIETARY FACTORS
`
`There are a numberof other factors that have been reported as essential
`in the diet of experimental animals, but to discuss them in any detail
`would be beyond the scope of this book. However, two definite chemical
`substances in addition to those already considered have been shown quite
`conclusively to belong to the vitamin B complex. These are para-amino-
`benzoic acid and inositol:
`
`Cc
`
`Coon
`a “cu
`|
`HCy. _CH
`|NH,
`
`Para-aminobenzoic acid
`
`“~i™
`
`#C
`Hoc # Bi
`BEIH HCOH
`Cc
`4
`Inositol
`
`The former is probably used for the biosynthesis of pteroylglutamiec acid
`and owes its vitamin-like activity in certain species to this circumstance.
`Inositol is required by mice andrats for normal growth and the avoidance
`of dermatitis and loss of hair.
`It is not known to be required by human
`beings.
`Another vitamin-like substance needed by certain microorganisms is
`lipoic acid, which has recently been obtained in pure form and found to
`have the following structure:
`Gerce(CH,),—COOH
`
`
`
`S
`5
`«-Lipoic acid or thioctic acid
`
`According to Reed and De Busk it is combined in the living cell with
`thiamine and phosphoric acid to form lipothiamide pyrophosphate, which
`appears to be a necessary coenzyme for the oxidative decarboxylation of
`a-keto acids, such as pyruvic acid, during metabolism.
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 258
`Petitioner Microsoft Corporation - Ex. 1032, p. 258
`
`

`

`VITAMINS
`
`bo wn on
`
`a
`(0=C—CH,CH.OP—0—P—OH
`Aa
`ae foe
`OH
`OH
`i—=G
`O=5Hi
`NH—CO(CH.)~CH—CHLCH,
`
`l
`s—5
`Lipothiamide pyrophosphate
`
`Still another compound, carnitine, has recently been shown by Carter
`and co-workers to function as a vitamin for a lower animal organism,
`namely,
`the larva of the yellow meal worm, Tenebrio molitor. These
`
`(CH,),N+CH.CHOHCH.COO—
`Carnitine
`
`the previously
`larvae will not grow on synthetic diets containing all
`known vitamins, but require the addition of supplements such as liver
`or whey. The effective substance was named vitamin By. When iso-
`
` a=
`
`: Sets
`es
`Courtesy of the 8. M. A. Corporation,
`
`Fig. 9-15. Pantothenic acid deficiency in the rat. These animals were
`reared on identical diets except that the one on the left received an ade-
`quate supply of pantothenic acid, while the diet of the other was deficient
`in this vitamin.
`:
`
`lated in pure form, it proved to be identical with carnitine, a compound
`which had long been known as a constituent of meat extract.
`It is
`possible that carnitine functions in the larvae as a source of labile methy!
`groups (p. 344).
`The so-called ‘“antigray-hair factor” may or may not be a definite
`substance different from the other known vitamins.
`It is well established
`that graying of the hair does result from certain nutritional deficiencies
`in various species of animals, particularly the rat, mouse, dog, and fox.
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 259
`Petitioner Microsoft Corporation - Ex. 1032, p. 259
`
`

`

`256
`
`VITAMINS
`
`Deficiencies of pantothenic acid, para-aminobenzoic acid, copper, and
`biotin have each been reported to cause such graying. However, there
`is at the present time no acceptable scientifie evidence that gray hair in
`human beings can be restored to its original color by the dictary use
`of any of these materials, or of any other “gray hair factor.’
`Otherless well-defined factors are vitamin P, which has been reported
`to correct bleeding caused by weakened capillaries in human beings,
`vitamin Bj,s, and vitamin By. A large numberof other vitamin-like sub-
`stances are apparently needed for the normal nutrition of various species
`of animals, and particularly of microorganisms, but knowledge of their
`nature and biological significance is too limited to warrant their con-
`sideration here.
`
`Antivitamins
`
`Substances chemically related to certain vitamins interfere with their
`normal physiological functioning and are therefore called antivitamins.
`For example, mice fed pyrithiamine (a thiamine analog, sce: formula)
`develop typical symptomsof thiamine deficiency.
`Similarly, pyridine-3-
`
`CH; CH,CH,OH
`
`CH
`
`CH;—C C—CH.—N
`i an
`=
`H
`.
`Pyrithiamine
`sulfonic acid and glucoa¥eorbie acid act as antagonists of nicotinie acid
`and vitamin C, respectively.
`In each case, administration of the vitamin
`concerned corrects the deficiency, and it appears that the response of
`the organism depends on the relative amount of the vitamin and anti-
`vitamin -present.
`An explanation for behavior cf this sort was advaneed by Woods and
`Fildes who found that p-aminobenzoic acid (PABA) can counteract the
`antibacterial effect of the drug, sulfanilamide. They suggested that
`PABAis an essential metabolite for the bacteria and that sulfanilamide
`exerts its effect. by acting as an inhibitor of the bacterial enzymes con-
`cerned with the metabolic us