VITAMIN G CON CENTRATES AS PREVENTIVES AGAINST
`
`BLACK-TONGUE
`
`WITH PARALLEL STUDIES on THE SAME CONCENTRATES IN THE DIETS or
`
`WHITE RATS
`
`L. E. BOOHER AND G. H. HANSMANN
`
`From the Department of Chemistrr , Columbia Unioersitr , New York and the Department
`of Pathology, Georgetown Unioerstti , Washington
`
`Received for publication September 6, 1935
`
`In 1927 Underhill and Mendel (1) described an extensive series of experi—
`ments dealing with a dietary deficiency disease in dogs which had previ-
`ously been described as pellagra—Iike by Chittenden and Underhill (2).
`The morbid anatomy of this canine disease was studied by Lambert and
`a report of his findings is included in the report of Underhill and Mendel.
`The morbid anatomy, as described by Lambert, agrees in the main
`so completely with the later report of the pathology of experimental
`black-tongue by Lillie (3) that there would seem to be little question that
`both were studying the identical canine disease.
`Goldberger and his associates, having been engaged in an extended
`investigation of the etiology of pellagra, had come to the conclusion that
`black—tongue in dogs was the analogue of pellagra in the human. This
`conclusion has been further substantiated by a comparative study of the
`tissue changes in experimental black-tongue of dogs and in pellagra as
`reported by Benton (4). A wide variety of natural food materials was
`investigated (5), under the auspices of the U. S. Public Health Service
`in continuation of the researches begun by Goldberger, for their relative
`richness in this pellagra—preventing or “P—P factor.’ ’
`Those foods which prevented pellagra were also efiective for the pre—_
`vention of experimental black—tongue. With the rapidly accumulating
`knowledge of the multiple nature of the “B—vitarnins” and their respec-
`tive distributions in natural foods, there was considerable indication, at
`least, that the “P—P factor” and vitamin G were identical.
`The vitamin G concentrate used in the investigation reported here
`for tests of black—tongue prevention and for parallel studies of growth—
`promotion in white rats carried both vitamin G (lactoflavin) and the “new
`heatwstable” factor or factors of the vitamin B-complex. The vitamin
`concentrate was prepared by a preliminary extraction of low-lactose whey
`powder‘with boiling 95 per cent ethyl alcohol, followed by concentration
`
`THORNE - EXHIBIT 1009
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`

`430
`
`L. E BOOHER AND G. H. HANSMANN
`
`drying. The details of the preparation have been described in an earlier
`report (6). This concentrate when supplied to young rats at a level of
`25 to 35 mgm. per rat per day with additional supplements of crystalline
`vitamin B (B1) as the only sources of factors of the vitamin B-complex
`induced a steady growth rate of 7 to 8 grams per week.
`The seven dogs used in control and black-tongue preventive tests were
`male dogs ranging from 6 to 10 lcgm. in body weight and were of mongrel
`types especially selected for unpigmented mucosa of the mouths. The
`dogs had every appearance of being in excellent physical condition at the
`beginning of the experiment. Dogs 1, 2 and 3 were given a conditioning
`diet (Sebrell (7) diet 326 as recommended by him) for four weeks before
`starting them on the experimental diet as an added factor of safety but
`this preliminary period was abandoned in the case of the other four dogs.
`During the course of the experiments the dogs were weighed weekly.
`Rectal temperatures were taken and examinations for lesions of the mouth
`were made daily. The practice of coprophagy was eliminated in all
`cases.
`
`Each of the dogs was given one of two black—tongue producing diets
`which were prepared essentially as recommended by Goldberger and his
`co—workers (8, 9) and which have been designated by them with laboratory
`numbers 123 (after some modification no. 268) and 324 respectively. We
`used a complete salt mixture (Osborne and Mendel salt-mixture with
`added copper sulfate) in each of the diets and included more cod liver
`oil in diet 324, neither of which modifications appears to have made any
`change in the production of black—tongue. The compositions of these
`black—tongue producing diets as used by us are as follows:
`
`grams
`White cornmeal ...................................................... 389
`
`Diet 1.931
`
`Cowpeas (California black-eyed) .....................................
`Casein (extracted with 60% alcohol) ..................................
`Sucrose ..............................................................
`Cottonseed oil .......................................................
`Cod—liver oil .........................................................
`Osborne and Mendel salt—mixture .....................................
`
`50
`66
`32
`30
`15
`24
`
`600
`
`Diet 3241
`
`grams
`White commeal ...................................................... 310
`Casein (extracted with 60% alcohol) .................................. 150
`Sucrose ..............................................................
`72
`Cottonseed oil. .' .....................................................
`26
`Coduliver oil ..........................................................
`31
`
`Osborne and Mendel salt-mixture ..................................... 3%
`613
`
`

`

`VITAMIN G AS PREVENTIVE OF BLACK-TONGUE
`
`4:31
`
`Each of these dietary formulas represents approximately a 2400 calorie
`quantity. Diet 123 contains 16 per cent and diet 324, 26 per cent of
`protein.
`The control dogs were fed daily quantities of one or the other of these
`diets equivalent to 100 calories per kilo of body weight. The allowances
`for the dogs receiving the vitamin concentrate were limited to 80 calories
`per kilo. The cornmeal and cowpeas used in these diets were purchased
`in single lots in sufficient quantities to last throughout the duration of the
`experiments.
`Two control dogs (dog 1 and dog 2) which received diet 123 showed
`definite prodromal symptoms involving the nervous system and gastro-
`intestinal tract after 19 and 18 days respectively, unmistakable mouth
`lesions after 33 days in each case and died after 37 and 38 days respec~
`tively.
`
`TABLE 1
`
`Summary of control dogs on black-tongue producing diets
`
`nos 1
`DOG 2
`
`not: 6
`
`
`
`nos 7
`
`324
`324
`123
`123
`Diet no ................................
`Prodromal symptoms became definite... 19th day 18th day 16th day 17th day
`Mouth lesions developed ............... 33rd day 33rd day 36th day 36th day
`Death occurred ........................ 37th day 38th day 56th day 38th“ day
`Total loss in weight“.................. 1.0 kgm. 1.8 kgm. 1.5 kgm. 1.6 kgm.
`
`* Dog developed grave central nervous system and gastro~intestinal symptoms
`and refused to eat after 32 days.
`** About 90 per cent of the loss in weight occurred during the last few days of
`life when there was anorexia, vomiting and diarrhea.
`
`Two control dogs (dog 6 and dog 7) which received diet 324 showed
`definite prodromal symptomsafter 16 and 17 days respectively, unmis—
`takable mouth lesions after 36 days in each case and died after 56 and 38
`days respectively. The general course of the decline was the same as that
`of the control dogs on diet 123 except that after the development of the
`lesions, the lesions progressed more slowly. Each of the control dogs
`lost weight during the test period.
`A summary of the response of the four control dogs is given in table 1.
`Dog 3, a 10—kgm. dog, was given diet 123 for 37 days at which time
`there had deveIOped vermillion spots on the mucous membrane of the
`mouth which were accompanied by gastro—intestinal symptoms. There-
`after the dog was furnished with 300 mgm. per kilo of body weight per
`day of the vitamin concentrate in addition to daily portions of diet 123
`corresponding to 80 calories per kilo of body weight. This dog was in
`
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`

`432
`
`L. E. BOOHER AND G. H. HANSMANN
`
`174 days after the experiment was begun and had gained two kilos in
`weight during this time. The daily doses of vitamin concentrate were
`reduced to 200 Ingm. per kilo per day after the first month of its admin.
`istration without any detectable change in the condition of the dog. The
`dog’s coat, like those of the other dogs receiving the Vitamin concentrate,
`became remarkably thick and glossy and the eyes very bright and ani-
`mated. The delay of 37 days before the administration of the vitamin
`concentrate accompanied by the signs and symptoms of black—tongue,
`as indicated, makes this experiment a more rigid test of the effectiveness
`of the vitamin concentrate than a strictly preventive test would haVe
`done. For all practical purposes it may be considered a successful cura-
`tive test.
`
`Dog 4, weighing 6.6 kilos, was given daily portions of diet 123 equiva-
`lent to 80 calories per kilo of body weight plus 300 mgm. of the vitamin
`concentrate per kilo of body weight from the beginning of the test. After
`one month diet 324 was substituted for diet 123. One month following
`this change the vitamin concentrate dosage was reduced to 200 mgm.
`.per kilo of body weight per day. This dog was in excellent condition at
`the termination of the experiment at 138 days and had gained 0.7 kilo
`in body weight during this period.
`Dog 5, weighing 7.1 kilos, was given daily portions of diet 324 in quan-
`tities corresponding to an 80 calorie intake per kilo of body weight for
`20 days by which time vermillion spots'had appeared on the mucous
`membrane of the mouth and gastro-intestinal symptoms had developed.
`Thereafter 300 mgm. of the vitamin concentrate per kilo of body weight
`were administered in addition to the basal ration, this quantity was subseu
`quently reduced to a 200 mgm. level without apparent change in the con—
`dition of the dog. This dog was in excellent physical condition at the end
`of the test period, 110 days after the beginning of the experiment and had
`gained 0.9 kilo in body weight since the administration of the vitamin
`concentrate was begun.
`In our opinion this concentrate is so clearly effective in creating the
`difference in the responses of the dogs on preventive or curative tests
`as compared with that of the control dogs as to make further extension
`in the duration of the experiments of no particular value.
`A summary of the responses of the dogs receiving the concentrate of
`the heat-stable factors of the vitamin B-complex are given in table 2.
`Twenty~eight day old white rats weighing 40 to 55 grams and of such
`nutritional background as we regularly employ for vitamin G researches
`were fed the black—tongue producing diets 123 and/or 324:. These diets
`were of the same composition as those given to the dogs.
`Six rats of each
`sex were given diet 123 ad libitum and six of each sex received the same
`
`

`

`VITAMIN G AS PREVENTIVE or BLACK-TONGUE
`
`433
`
`which were used for the tests of black—tongue prevention in dogs. Simi-
`larly, an equal number of each sex was given diet 324 alone and a further
`group diet 324 plus 40 mgm. per day of the vitamin concentrate. The
`
`TABLE 2
`
`Su'imnary of dogs resetting black—tongue producing} diets supplemented with vitamin G'
`concentrate
`
`‘ D
`
`DOG 5
`Doc 4
`oc 3
`
`Diet no ........................
`123
`123 and 324*
`324
`Quantity of vitamin G concen— 300 mgm. 30th
`300 mgm. first
`300 mgm. 20th
`trate given per kgm. per day
`to 60th day,
`to 60th day,
`to 60th day,
`200
`mgm.
`200
`mgm.
`200
`mgm.
`thereafter
`thereafter
`thereafter
`176 days
`138 days
`110 days
`Duration of experiment .........
`2.0 kgm.
`0.7 kgm.
`0.9 kgm.
`Total gain in weight ............
`
`
`ExcellentExcellentGeneral physical condition ...... Excellent
`
`
`* Diet 123 was given for the first 30 days, diet 324 thereafter.
`
`
`
`Fig. 2
`Fig. I
`Fig. 1. Curves I and 2 summarize the average growth rates of six young female
`and six young male rats, respectively, on black-tongue producing diet 123. Curves 3
`and 4 are littermate control (female and male respectively) rats which received
`diet 123 plus 40 mgm. per day of the vitamin G concentrate.
`
`Fig. 2. Curves I and .2 summarize the average growth rates of six young female
`and six young male rats, respectively, on black-tongue producing diet 324. Curves 3
`and 4 are littermate controls of rats represented in curves 1' and 2 but which received
`in addition to diet 324, 40 mgm. per day of the vitamin G concentrate.
`
`growth rates of these four groups of rats on the black-tongue producing
`diets 123 and 324 with and without administration of the vitamin concen-
`
`

`

`434:
`
`L. E. BOOHER AND G. H. HANSMANN
`
`case grew at sufficiently different rates to Warrant separate plotting of
`their growth rates. The rats representing these four groups were litter-
`mate controls. The practice of coprophagy was eliminated in these tests.
`As is evident from these curves, the rats receiving diet 123 alone grew at
`a greater rate than those receiving diet 324; alone. However with the
`addition of equal amounts (40 mgm. per day) of the vitamin concentrate to
`these diets the rats on diet 123 grew less than those on diet 324:. Neither
`of these diets is entirely devoid of the heat—stable factors of the vitamin
`B-complex as judged by these experiments since the rats receiving either
`diet 123 alone 01' diet 324 alone grew at slow rates. The growth rates,
`however, were much augmented by the addition of the vitamin concen-
`trate.
`
`We believe that the nutritional superiority of diet 324 in respects other
`than its vitamin G content as compared with diet 123, as shown in the
`experiments both with rats and with dogs, may be largely attributed to
`the higher protein content of diet 324:. This nutritional difference be~
`tween the two black-tongue producing diets supports the earlier indication
`of Goldberger and his associates that pellagra cases were benefited by the
`addition of protein to their diets even though the “P-P factor” was later
`found to be the specific dietary deficiency. This nutritional interrelation
`of protein and vitamin G has also been affirmed by Sherman and Derbigny
`(10) in experiments with rats.
`DISCUSSION. The most distinctive lesion of black-tongue is observed
`in the mucosa of the mouth. This lesion, however, occurs rather late in
`the progress of the disease and appears as reddened patches on the mucosa
`'of the upper-lip, or of the check or floor of the mouth. With further
`progress of the disease there appear superficial necrotic areas within these
`reddened patches.
`It is probably because these lesions are so distinctive
`of the disease that the more disturbing prodromal symptoms involving
`the central nervous system and the gastro-intestinal tract have not been
`more strongly emphasized. Black—tongue should be regarded as a disease
`of severe systemic disturbances rather than a disease merely of severe
`buccal lesions. Epithelium and cells derived from epithelium embryo-
`logically are the elements of structure which suffer severely in black-
`tongue.
`.
`Our experiments, thus far, do not indicate whether only one or more
`than one of the heat—stable factors of the vitamin B—complex is involved
`in the prevention or cure of black—tongue. The parallelism in growth
`stimulating properties of the vitamin concentrate for rats and the black-
`tongue preventive or curative properties of this same concentrate for
`dogs would indicate that an identical substance (or substances) carried
`by this concentrate was the effective agent for promoting these respective
`
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`

`VITAMIN G as PREVENTIVE or BLACK-TONGUE
`
`435
`
`B~complex, when given as a supplement to young rats on either one of
`the black—tongue producing diets, will augment growth.
`The used 200 mgm. of our vitamin concentrate per kilo of body weight
`of the dogs appears to allow for a considerable margin of safety since
`there was every indication that our treatment dogs were generously sup—
`plied with the black-tongue preventive factor.
`The early manifestations in the control dogs which indicated a defi-
`ciency of the black-tongue preve'ntiVe factor, whether or not the dogs
`had received the liver diet. (conditioning diet) in a preliminary period,
`would indicate that the bodily stores of this factor were insufficient for the
`maintenance of health for longer than sixteen to nineteen days. The
`practical implications of this rapid depletion of bodily stores of black-
`tongue preventive factor should not be overlooked.
`
`-
`
`SUMMARY
`
`A vitamin G concentrate obtained from low~1actose whey powder
`which carries, in addition to vitamin G (lactoflavin), at least one other
`heat—stable vitamin necessary for rat-growth, has been found effective
`for the prevention or cure of black—tongt‘re. A daily quantity of 200 mgm.
`of this vitamin concentrate per kilo of body weight per day was more
`than sufficient to protect dogs against black-tongue.
`The diets used for the production of black-tongue in dogs induced an
`inferior rate of growth when fed to young rats. These growth rates were
`augmented as a result of supplementing these diets with the vitamin
`concentrate which was preventive or curative for black—tongue.
`There is evidence that the inclusion of liberal quantities of protein of
`good nutritive value decreases the vitamin G (and/or other heat-stable
`vitamins) requirements for the growth of young rats and slorvs up the
`progress of the development of the mouth lesions of experimental black-
`tongue.
`'
`
`We wish to express our thanks to the American Medical Association,
`Council of Pharmacy and Chemistry for a grant—in—aid of this research.
`
`REFERENCES
`
`(1) UNDERHILL, F. P. AND L. B. MENDEL. This Journal 83: 589, 1927.
`(2) CHITTENDEN, R. H. AND F. P. UNDERHILL. This Journal 44: 13, 1917.
`(3) LILLIE, R. D. National Inst. of Health Bull. 162: 13, 1933.
`(4) BENTON, J. Am. J. Path. 4: 341, 1928.
`(5) GOLDBERGER, J., G. A. WHEELER, R. D. LILLIIL‘ AND L. M. Rooms. Pub.
`Health Rep. 43: 1385, 1928; also G. A.'WHEELER AND W.- H. SEBRELL.
`Nat. Inst. of Health Bull. 162: 1, 1933.
`(6) Boonnn, L. E.
`J. Biol. Chem. 102: 39, 1933.
`
`