vn'ama’s
`
`241
`
`occurs in two forms, dextrorotatory and levoi':itator_\', and of these only
`the dextrerotatory form has biological activity.
`time free pantothenie
`acid can readily be obtained only as a sirupy. gummy mass, it is usually
`converted to the calcium salt, which is a white powder and the form in
`which the synthetic product is supplied.
`Since it is an amide, pantothcnie acid is readily hydrolyzed by heating
`in either acid or alkaline solution. Hydrolysis results in complete de-
`struction of the vitamin activity.
`It is rather stable to boiling in neutral
`aqueous solutions, although it is destroyed by long heating at 120%).
`It appears that pantothenic acid is not‘ extensively destroyed by ordi-
`nary cooking of food. Losses of approximately 50 per cent may, however,
`occur if the cooking vater from vegetables is discarded.
`
`Occurrence
`
`liver, egg yolk, and rice polishings are very rich sources ol‘
`Yeast,
`pantothenic 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 pantotlienic 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 pantotlicnie acid has not yet been determined,
`but it has been suggested that about 10 mg. per day is adequate.
`
`PYRIDOXINE (VITAMIN Be)
`
`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 nose are 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 nia-
`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
`
`

`

`3-12
`
`VITAMINS
`
`in \‘:l1"ll!tl.-5 animals include.- a type of anvmia and tits
`lit-0n envnuntt-rutl
`resembling epilt'ptic seizures in human beings.
`l’yridnxine tlefivibncy in
`man has hurt: uhsorwd in a number of (mars of pellngrins who still were
`nut lrmnplutt-ly “I'll after I'Ct-t'iving nitmtinit' acitl. thiamine anti riboflavin.
`Hyunm-ms nnml in Sllt'll pativnts were nervmismlss, irritability, abdominal
`pain. wallinuss, and difficulty in walking. Tln'sv sylliptuins were quickly
`rulicvt‘cl by tllt‘ lh-‘t'
`(Jl~ synthetic pyridnxint’.
`
`Chem frat nature
`
`Pyrithixinv, or Vitamin B“, was first isolated as a pure chemical sub-
`stance in 1938, and during the next year it. was. Inn-pared synthetically.
`
`r._..__w.;
`
`._
`
`_w..._,.__..
`
`
`
`L'ourlwy of Merck & ‘70..
`
`Inc.
`
`Fig. 9—12.
`
`l’yritluxinv.
`
`The chemical nature of this vitamin is best expressed by its structural
`formula:
`
`CH20H
`
`|C
`
`/ “H.
`
`HO—Clics)m?g)t£|I—CH20H
`(2)
`5
`CH,-—cxcfigcn
`Pyridoxine, CsHuNOs
`
`Note that it is related to nicotinic acid in that it is a pyridine derivative.
`The name pyridoxinc is derived from the chemical munc for this substance,
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 246
`Petitioner Microsoft Corporation - Ex. 1032, p. 246
`
`

`

`VITAMINS
`
`243
`
`which is 2-Iuethyl-3-liytti'oxy-4,;')-di-lihydl'oxymetllyll-pyrirline.
`Two closely related substances, pyridoxal and pyridoxamine, are l’epl‘t‘»
`sented by the following formulas:
`
`CHO
`i
`Honel’ *e—cmon
`HaC—C\N¢CII
`I’yridoxal, Cngoai'
`
`CHaNH:
`a;
`HO—(fi’ se—cnpn
`liaC—C‘NfiCH
`Pyridoxamine, 031:1:03N ._.
`
`rl‘tlese substances have about the same. vitamin 15.; activity for animals and
`for yeast cells as pyridoxine does, but are several tlmusand times more
`effective for certain bacteria. A plmsphorylated derivative, pyridoxat
`phosphate, functions as a eocnzyme for enzyme systems present in many
`bacteria, which break down amino acids into the corresponding,1r amines
`by removing carbon dioxide from the earboxyl group of the. amino acid
`in. 321).
`It is therefore called :1 eodeearboxylase. Both pyridoxal phos-
`phate and pyridoxamine phosphate function as eoenaymes in certain trans-
`amination reactions (p. 343) and may, therefore, be called eotransami-
`oases.
`
`CHO
`3:
`lie—(l? ‘fi—cnio—Poan
`HaC—C‘sNfiI—I
`Pyridoxal phosphate
`
`CH :NH:
`(‘3
`HO—JTV F—CHp—Posn,
`H3C—CsN,CH
`Pyridoxamine phosphate
`
`Pyridoxal phosphate also serves as a eoenzyme for the enzyme system
`involved in the synthesis of tryptophan by a certain mold species (Neuro-
`spora erased).
`It is,
`therefore, quite clear that the 13;; vitamins play
`important roles in both the decomposition, interconversion, and synthesis
`of amino acids in living cells.
`Pyridoxine is stable to heat, alkalies, and strong acids, but is rather
`easily attacked by oxidizing agents. As yet little “'Ul'k has been done
`on its destruction during the cooking of food. No reliable figure for the
`human requirement is available, but a tentative value of 1.5 mg. per
`day has been suggested.
`
`Occurrence
`
`Pyridoxine is present in yeast, bran and embryo of 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
`
`I'yridoxine content of common foods
`
`......................................
`.
`.
`.
`.
`.
`.
`.
`Beef, lean .
`Beef. liver ................................................
`Breatl.\vliiic.....__..._._..................................
`Bread. whole wheat
`. ......................................
`Cabbage. ............................................... .
`C'urrot ......................................................
`Chicken, dark meat .
`.
`......................................
`Lamb. leg of .................................................
`Milk. whole
`i ...............................................
`Oatmeal ...................................................
`Pork loin .................................. _..................
`Potatoes, white ..............................................
`Yeast. dried brewer’s . ........................................
`
`illiltigroms per 100 y.
`edible portion
`0.40
`0.73
`0.30
`0.70
`0.29
`0.19
`0.20
`0.38
`0.20
`025
`0.60
`0.16
`5.5
`
`BIOTIN
`
`This member of the vitamin B complex is a substance which has been
`variously known as “coenzyme R,” “vitamin ll," “biotin,” and the “anti~
`egg white injury factor.”
`It was first obtained in pure form and given
`the name biotin in 1936 by Keg], who was studying it as one of the
`vitamindike 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, acidic,
`present in raw egg white. When so combined, biotin cannot be absorbed
`and utilized by the animal organism. Cooked egg white on the other
`hand is perfectly 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 produce the “egg white injury” dis-
`ease 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 deficiency has been
`observed in human beings consuming their customary diets. The daily
`intake of biotin on an average diet ranges from 25 to 50 he,” and the
`urine and feces together may contain from two to five times these
`quantities.
`
`
`
`l‘nnrhnsy of the H. M. A. Corpnrnlioll.
`
`Fig. 9—13.
`
`ltioliu.
`
`Biotin appears to function [possibly in the form of a coenzyine, al-
`though none has yet been identified) as a catalyst for one of the reac-
`tions of the citric acid cycle ([1. 330):
`
`002 + CHa—CO—COOH —) HOOCTCHg—C0——COOH
`Pyruvic acid
`Oxalacctie acid
`
`Further, certain lactobacilli which normally require biotin grow well with-
`out it if oleic 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 oIeie acid. The vitamin is
`also required for deamination by bacterial cells of serine, threoninc, 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 structure in one of the rings (A) and the
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 249
`Petitioner Microsoft Corporation - Ex. 1032, p. 249
`
`
`
`

`

`346
`
`VITAMINS
`
`presence of sulfur in the other ring (13}. Biotin and thiamine are the only.r
`vitamins that contain sulfur.
`
`Biotin, ClochoaNgs
`
`Although it is readily destroyed by such oxidizing agents as hydrogen
`peroxide, biotin is, in general, a ver}r stable substance.
`It is not affected
`by light, strong acids such as normal HC-l or Hut-104, nor by exposure to a
`degree of heat greater than that encountered during ordinary cooking
`operations. However, it is destroyed by strong alkaii. , In mamr tissues
`it appears not to 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 bioeytin, a peptide-like combination
`of biotin and the amino acid, lysine. Note that the linkage is through
`the epsilon amino group of lysine.
`
`ti")
`o
`HN/ \‘NH
`|
`HC—HCH
`!
`|
`
`S
`
`NH .
`
`Biocytin
`
`Nothing is knorvn 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 client 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 requirements of lactic acid bacteria. An impure preparation from
`liver, designated as the “norite eluate factor,” was Shawn to be necessary,
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 250
`Petitioner Microsoft Corporation - EX. 1032, p. 250
`
`

`

`VITAMIN-s
`
`247
`
`in addition to previously known vitamins. for the normal growth of these
`organisms. The efTective substance present
`in such preparations was
`later found to be identical with “factor [7” 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 he pteroylglutamie acid,
`or closely related compounds. These names included “vitamin BC,"
`“factor R," “factor S,” “folic acid,” “8.
`tor-[is R factor or SLR factor,”
`“liver L. casei factor.” The term folic acid is still in use, but should
`
`now be replaced by the proper chemical names {see below}.
`
`Chemical nature
`
`Pteroylglutainic acid is a complex substance made up of three parts,
`glutamic acid, para-aminohenzoic acid (p. 254], and a pterin, chemically
`linked together:
`
`N
`
`H.
`0
`
`ncgfi’tfifi—conHelncnamfioon
`Haz—(lig/lll‘filf/‘Sfigtlcn
`NQg/cc‘tlfli/e—cmnn——cSaigon
`COO“
`
`I
`OH
`
`H
`
`Ptcroylglutamic acid
`
`This substance is identical with the “liver L. easei factor,” vitamin B0,
`and iolic acid. The name jolocin was proposed in 1949 by the American
`Institute of Nutrition as a synonym for folic acid. The “fermentation
`L. coset' factor” is very similarly constituted except that three glutamic
`acid residues are present.
`In this form, also called teropterr'n, the second
`and third glutamie acid residues are linked to the preceding one through
`the gamma carboxyl group rather than through the alpha carboxyl {see
`p. 131). 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 rhizoptertn, contains no glutamic acid
`at all but bears an aldehyde or formyl group on the nitrogen atom in
`position 10:
`
`H H
`/N Nx /
`CHO 0—0
`H ,N—q/{ll C {3):‘CH
`I
`//
`\\
`If;
`H
`”
`NQ(4&/C\(5)9C—qu—g§)—C
`/c—coon
`N/
`
`I
`OH
`
`HC=CH
`
`Formylpteroic acid (or rhizopterin)
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 251
`Petitioner Microsoft Corporation - Ex. 1032, p. 251
`
`

`

`243
`
`VITAMINS
`
`The glutamie acid derivative of rhizopterin, formylpteroylglutamic acid,
`or formyl folie acid, has been prepared synthetically and found to possess
`the typical vitamin activity of other members of this group.
`It probably
`also occurs naturally. Rhimptcrin itself, however, does not relieve the
`symptoms of folic acid deficiency in higher animals.
`Very recently a substance needed for normal growth of the bacterium
`Leuconostoc ca'troeorum (the so-called “citrovm‘um factor”) has been
`found to be closely related to formylpteroylglntamie acid, from which
`it can be obtained by reducing and heating. The product, named foli'm'c
`acid by one group of investigators and teacowrin by another, has been
`shown (Consulieh, et (11., l’ehland, et at.) to have the following formula:
`
`11
`N
`
`N
`
`H
`
`n
`
`econ
`;_c{\
`Has—(l3¢ “E” “(EH2
`Na“ /0\ yc—cngna—d
`lc—conacncaicnnoon
`cf
`1?
`C=o
`on
`CHO
`H
`H
`Folinic acid (or lcucovorin)
`
`In many tests 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 pteroylglutamic acid itself.
`
`Physiological function
`
`This vitamin is essential for a wide variety of living organisms, and,
`in fact, is probably needed by all 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 human diseases are the result of a lack of pt-eroylglutamie
`acid or related substances is indicated by the improvement which follows
`their administration. The best example is sprue. a disease characterized
`by maerocytic 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 ptemylglutamic acid or of the triglutamate,
`teropterin, result in prompt relief of these symptoms. Related conditions
`described as nutritional macrocytic anemia and macrocytic anemia of
`pregnancy are similarly benefited. Pernieious anemia patients are bene-
`fited somewhat, but the improvement is temporary and incomplete,
`in
`contrast to the effects of vitamin B12 {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 arhuinistration 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 eo111ponents
`of nucleic aeids.Te10pte1in has been cl 111imtd to 1(1Ii1'1e pain in advanced
`cases of human cancer and to 1eta1'd the growth of t11111o1s in experimental
`animals ..
`,1,
`
`Food sources and! requirements
`
`u".
`
`-
`
`1-
`
`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 oeeur1 ed dining canning When a solution of the
`pure vitamin was placed1n b11ght daylight for 8 hours, 88 per cent 1111s
`destioy ed.
`:31
`e
`,According to Toepfel and co-workers a nuinbei of common foods may
`beEscaped as follons, o1'i-'tl1e basis of the milligrams of folic acid which
`they contain per 100 g. of city 11eight: 01'e1 19,:b1e11crs yeast, chicken
`liver,3sparagus, broadleaf endiv,e lJIOCCOll, lefiyflettuce, spinach; 0.4—1.,0
`most of the other leafy greens,
`li1er, blackeve peas, dried beans soy
`flour; 01—64, other vegetables except root 1egctables and a few fruits;
`0.03—0.l,1tiot vegetables, most fresh fruits, glains 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 3,2
`
`It has long been recognized that liver and suitable extracts prepared
`from liver contain some substance which is 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-
`nieious anemia factor” in liver have been made. With the discovery
`of pteroylglutamie acid and the observation that it is effective in caring
`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 8,2,
`contained 4.4 per cent cobalt, 2.3 per cent phosphorus and had the formula
`CGI_G4IIS.;_92N14013PCU (molecular weight about 1350). Although the
`
`’
`
`fl
`
`‘
`
`fl —
`f
`
` ‘
`
`j.
`
`4
`
`.I“
`
`s...
`
`__
`
`Courtesy of
`Fig. 9-14. Vitamin Br.‘ crystals (X 200).
`
`‘W.
`.-\hliotl: Laboratories.
`
`complete structure is not yet. known, several fragments of the molecule,
`including 5,6-di1nethy1 benzimidazole, have been identified after acid
`hydrolysis. Surprisingly, this substance itself showed full vitamin B13
`activity for rat growth when tested in 5 mg. daily doses. Other hydrolysis
`
`Hc
`
`Hacc¢ “c/\
`Ha,
`ll
`0..
`a
`\C/ \N
`H
`H
`
`5,6-Dimethyl benzimidazole
`
`products identified are propanolamine (CIIgCHOHCHgNHg) and a phos-
`phorylated derivative of the 5,6-diinethy1benzimidazole {ribcse-3-phos-
`phate attached to the N at. position 1). Vitamin B“; also contains 3
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 254
`Petitioner Microsoft Corporation - Ex. 1032, p. 254
`
`

`

`VITAMINS
`
`25]
`
`cyanide group (CNJ bound in a coordimition complex with the cobalt
`atom, which can be replaced by C], EU.“ Uli, SUN, or other groups to
`produce analogs of the natural substance. The analog containing the
`water molecule has been called vitamin It”, and is apparently identical
`with another preparation provisionally designated B12». Brink and co-
`workers have suggested that the B1: molecule, except
`for the cyanide
`group, be called cobalmm’a. By this nomenclature, vitamin 13,2 would
`be named cynno-cobnlamin and B12“, hydrate-cobalt:min. All of these
`’arious forms of the vitamin have approximately the same lsind and
`amount of biological activity.
`
`Physiological function
`
`In the short period since its isolation vitamin B12 has acquired excep-
`tional practical
`importance because of
`its demonstrated usefulness in
`pernicious anemia and related diseases, in livestock feeding, and in launan
`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 month because pernicious anemia patients lack
`some substance (“intrinsic factor”)
`in the gastric juice which protects
`vitamin B13 and favors its absorption. Small doses of vitamin 131-; are
`also effective in sprue and other macrocytic ancmias.
`Sec Plate IV op-
`posite p. 223.
`It has been known for many years that animal protein supplements
`(8.9., meat scraps, dried whey, etc.) 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 {APFJ. Vitamin
`B12 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 B12 has made possible 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 B12 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 B12 concentrates from these sources gave
`greater growth responses in some species than could be accounted for by
`their 1313 content. The extra effect was traced to the antibiotics still
`
`present as impurities in the concentrates. This discovery has opened
`new vistas 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
`
`

`

`251’
`
`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 Wotzcl et al. have reported that doses of 10 ,ag. of vita-
`min B}: given daily by mouth to a group of malnourished school children
`resulted in definite stimulation of growth in :3 of the 11 cases treated.
`These results establish the existence of human vitamin B12 deficiency
`other than that of pernicious anemia. How extensive this may he remains
`to be. determined by further study, but present indications are that vita-
`min Bu may well prove to have wide applications in human nutrition.
`The metabolic function of vitamin B12 in the animal body is evidentiy
`closely related to that of pteroylglutamic acid (for example, both are
`effective. in certain types of anemia). Specifically, vitamin B12 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 homoeystinc}.
`
`Food sources and requirements
`
`As already indicated, vitamin Bu 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 Eivehjeln 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 Bu has been established, but
`1 pg. per day,
`if injected,
`is sufficient
`to maintain pernicious anemia
`patients in good condition. This amount is much less than the minimum
`human requirement, of any other vitamin or trace element.
`Table 9—6
`
`Vitamin Bu contenl of foods
`
`(Micrograms per 100 g., fresh basis)
`Minimum vitamin
`‘
`Bu content
`Fooo
`*
`Egg yolk ..... . ......
`*
`Goat's milk .....
`_
`.
`.
`Green pens ...... .
`.
`_
`Horse meat, canned .
`.
`Mutton .
`.
`........
`Pork, shoulder .......
`Pork, ham ..........
`Potatoes .
`.
`.........
`Toronto juice ........
`Veal ................
`
`FOOD
`Barley ..............
`Beans ...........
`.
`Beef,liver ...........
`Beef, kidney .........
`Beef, round, cooked.
`Beef, tongue .
`.
`.
`.
`.
`.
`.
`.
`Cabbage ..........
`Cheddar cheese .
`.
`.
`Chicken liver .......
`.
`Cow’s milk .
`.
`.
`.
`.
`.
`.
`.
`.
`* No measareable amount.
`
`.
`
`15
`20
`2—3
`3
`*
`1.4
`11
`Trace
`
`Minimum vitamin
`B12 content
`1.4
`*
`*
`3.4
`3
`1.1—2
`1.2
`*
`"‘
`2
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 256
`Petitioner Microsoft Corporation - EX. 1032, p. 256
`
`

`

`vrmmrx‘s
`
`CHOLINE
`
`1.53
`
`Physiological function
`
`A lack of choline in the diet 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. 0n the other hand, feeding choles-
`terol aggravates the condition. Older rats are much less 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 mas
`tcrials) in the liver. The evidence at present available is consistent with
`the assumption that neutral fat [that is. glyecrides) must be converted
`into phospholipides before it is transported elsewhere in the body or, if
`it remains in the liver, before it
`is catabolizcd. Since choline is one
`component of the lecithin type of phospholipides, it would obviously be
`needed for these purposes.
`In fact, 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 pcrosis, in which the leg:r tendon
`slips off from the heck 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-
`scribed in Chap. 13.
`
`Chemical "more
`
`Choline is a very strong base, with the following structural formula:
`
`/CHs
`HOCH;-- CH 2—Iilq—CH;
`HO OH:
`
`Choline
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 257
`Petitioner Microsoft Corporation - EX. 1032, p. 257
`
`

`

`254
`
`VITAMINS
`
`Like nieotinic 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
`diiute aqueous solution. Hot alkalies, however, decompose it with the
`formation of trimethylamine.
`Bound choline in the form of lecithin is present in may living cell,
`and free choline is likewise very widely distributed in biological materials.
`At present. no information is available regarding the human requirement
`for this dietary factor.
`
`OTHER DIETARY FACTORS
`
`There are a number of 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-
`benzoie acid and inositol:
`
`(130011
`C
`ml}? ““03
`II
`CH
`\‘0/
`I
`NH:
`Para—aminobenzoic acid
`
`HC
`
`/ \
`
`3C
`HOUR H HCOH
`noon
`HCOH
`\Jé/
`0
`H
`Inositol
`
`The former is probably used for the biosynthcsis of pteroylgiutamic acid
`and owes its vitamin-like activity in certain species to this circumstance.
`Inositol is required by mice and rats 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
`lipoie acid, which has recently been obtained in pure form and found to
`have the following structure:
`
`(EH-201123311(CH1){-COOH
`
`S
`S
`
`a-Lipoic acid or thioctic acid
`
`According to Reed and De Busk it is combined in the iiving cell with
`thiamine and phosphoric acid to form lipothiomfde pyrophosphate, which
`appears to be a necessary coenzyme for the oxidativc 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
`
`

`

`\‘I’l‘.-\ 3113\5
`
`255
`
`ens
`
`o
`
`o
`
`XLII=C~CI13CILOii—-O—~ii’— on
`lib?”
`EGO—(i
`fi_CH“_§\\
`(in
`(in
`NH?
`fi—S
`NH—CO(CELL—CH—Cllx‘ltq
`é_é
`Lino thiamidc pyrophosphate
`
`Still another emnpouml. eal'nitinc. has recently been shown hy Carter
`and ell-Workers to iunetion as a vitamin for a lower animal organism,
`
`namely,
`
`the larva of the yellow meal worn], Tenebrio moh'foi'. These
`
`(CII;.i_...\' ~r (‘ngr‘i [onentt ‘oo—
`Caruitinc
`
`the previously
`larvae will not grow on synthetic- diets containing all
`known vitamins,
`liut require the addition of supplements such as liver
`or whey. The effective substance was named vitamin B-p. When iso-
`
`
`
`Courlesy of the S. M. A. Corporation.
`
`'l'hese. animals were
`Fig. 9—15. Pantothcnic acid deficiency in the rat.
`reared on identical diets except.
`that. [he one on lhe lel't received an ade-
`quate supply of pantothenic acid, while the diet of the other was deficient
`in this vitamin.
`'
`
`latcd in pure form, it proved to be identical with carnitinc, a compound
`which had long been known as a eonstilucnt of meat extract.
`It is
`possible that carnitine functions in the larvae as a source of labile methyl
`groups (p. 344].
`The so-cnllcd “antigray-hair factor" may or may not he a definite
`substance different from the. other known vitamins.
`It is Twell established
`
`that graying of the hair does result from certain nutritional deficiencies
`in venous 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
`
`vITAMle
`
`Deficiencies of pantothcnic acid, para—aminobenzoie acid, copper, and
`biotin have. each bccn reported to cause such graying. However, there
`is at the present time no acceptable Scientific evidence that gray hair in
`human beings can be restored to its original Color by the dietary use
`of any of these materials, or of any other “gray hair factor.”
`Other less well-defined factors are vitamin P, which has been reported
`to correct bleeding causcd by weakened capillaries in human beings,
`vitamin B13, and vitamin B“. A large number of 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.
`
`Antivilamins
`
`Substances chemically related to certain vitamins interfere with their
`normal physiological functioning and are therefore called antivitonn'ns.
`For example, mice fed pyrithiaminc (a thiamine analog, see- formula)
`develop typical symptoms of thiamine deficiency.
`Similarly, pyridine-3-
`
`(IJHa CH,CH,0H
`M-
`CH
`CH.—C C—CHr-N
`II
`II
`+\
`(J/
`N—CH
`fi=H
`Pyrithiamine
`-
`sulfonie acid and glucoascoi'bie acid act as antagonists of nicotinie acid
`and vitamin C, respectively.
`In each case, administration of the