By Cyrill William Carter
Summary: An important article about one of the critical B complex vitamins that got lost in the rush to synthesize nutrients. Vitamin B4 is a vitamer of the B complex that promotes proper nerve impulse transmission, yet it is not recognized as an essential nutrient by modern science. In the report Oxford researcher Cyrill William Carter notes that in pigeons suffering heart block who had been fed a diet devoid of natural vitamin B complex, supplementation with vitamins B1 and B2 failed to resolve the problem. When supplementation was switched to a yeast extract, which naturally contained the then-unknown B4 vitamer in addition to vitamers B1 and B2, the heart block was resolved. Oxford University scientists worked for over a decade to resolve the relationship between vitamin B4 and vitamin B1. From the Biochemical Journal, 1934. Lee Foundation for Nutritional Research reprint 3.
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Maintenance Nutrition in the Pigeon and Its Relation to Heart Block
In a previous paper (Carter, 1930), it was shown that pigeon heart block that develops on a diet of polished rice could be cured if the birds received daily rations of whole wheat constituting not less than about 60 percent of the total food intake. Vitamins B1 and B2 and the fat-soluble group of vitamins were found to be ineffective in restoring normal heart function when administered as supplements to the diet of polished rice. Heart block in the pigeon is therefore a condition of dietary origin. The question remains whether it is due to the deficiency of a specific factor, as is the case in polyneuritis, or whether it arises from a deficiency of one or more of those factors that contribute to maintenance nutrition at maximum weight.
In some experiments, to be described, it will be shown that pigeons previously depleted on a diet of polished rice can, in a majority of cases, be restored to maximum weight by [adding] appropriate supplements to this diet without inducing any recovery of normal heart function. Substitution of an extract of hydrolyzed wheat germ for the supplements results in a complete recovery from heart block without further significant change in weight. It will be further shown that birds previously brought to maximum weight on whole wheat can be maintained at this level on a diet that fails to protect them from the onset of heart block.
The fact, previously recorded, that birds depleted on polished rice, when fed on whole wheat, generally recover normal heart rhythm at a time when restoration of body weight is far from complete has been confirmed. Finally, the protein intake of birds on polished rice diet has been examined, and its significance in relation to the problem of pigeon nutrition is considered.
Birds under test were kept singly in small cages in order to obtain measurements of the food intake. Observations on body weight were made at the same time each morning in order to avoid, as far as possible, fluctuations due to variations in the amount of food present in the crop. Heart rate and rhythm were noted at frequent intervals.
The source of vitamin B employed was a yeast concentrate prepared according to the method of Kinnersley and Peters (1927) by the extraction of activated norite charcoal with 50 percent acid alcohol. (This partially purified concentrate may contain, in addition to vitamin B1, another factor, vitamin B6, which has been suggested as being necessary for the pigeon (Carter et al., 1930).) In all cases cod-liver oil was given at regular intervals.
Caseinogen [casein] (Glaxo, “Vitamin B-free”) was extracted continuously in a Soxhlet apparatus with 90 percent alcohol for 48 hours and then dried. It has been assumed in this paper that caseinogen treated in this manner provides a source of protein free from possible contaminating substances that might influence the nutrition of the pigeon. Before administration by pipette, portions of 2 grams were weighed out, suspended in 20 mL Ringer solution, and brought to pH 7.0 by addition of caustic soda.
Hydrolyzed wheat germ was prepared by heating for 3 hours with N-hydrochloric acid and filtering hot on a Buchner funnel. This method, which differs from that employed by O’Brien (1934), was used daily to prepare the extract, in order to avoid the possibility of its deterioration on standing in air.
The earlier experience of the effect of marmite—which daily 1.5-gram doses of, administered over periods of 30 days and upwards, showed curative activity in regard to heart block in a certain proportion of cases—prompted a hope that the factor concerned might be fractionated from yeast. The negative results that have invariably been obtained with vitamin B concentrate suggested that preparations from the precipitate that appears on treatment of an aqueous yeast extract with basic lead acetate might be effective. The results have unfortunately been variable and do not at present justify detailed consideration.
The lead precipitate, after treatment with sulfuric acid followed by hydrogen sulfide to remove all traces of lead, when administered to pigeons fed on polished rice, is entirely without influence either in preventing decline in weight or in improving the heart condition. In an experiment in which this extract was supplemented by daily doses of an antineuritic concentrate together with 2 grams caseinogen, two birds showed restoration of normal heart function within 20 days, while in another bird definite improvement, though incomplete cure, was noted. In all three cases, there was gradual recovery, over a period of 50 days, to maximum weight. Repetition of this experiment with similar treatment resulted in complete cure of heart block in one bird, partial recovery in a second, and no improvement in a third. Restoration to weight maximum was observed in two of these birds, while in the third the rise of weight, though substantial, failed to reach that obtained on whole wheat.
These preliminary experiments made it important to ascertain whether the induction and cure of heart block could be dissociated from factors associated with maintenance or restoration of normal body weight.
Effect of Yeast Concentrate and Caseinogen on Pigeon Nutrition
A group of eight birds was placed on a diet of polished rice for periods varying from 12 to 24 days. During this time the usual fall of weight and onset of heart block occurred. In one case acute polyneuritis developed before treatment was commenced. There is at present insufficient evidence to indicate whether variation in the duration of the depletion period has any influence on the subsequent response. A control group of three birds was given daily doses of a vitamin B1 concentrate in amounts equivalent to 12 units daily.
Table I and Figure 1 show that the progressive decline in weight is arrested and that there is some rise which is less marked than that observed with birds receiving in addition a daily supplement of 2 grams caseinogen. This confirms previous experiments in which maximum weight has not been fully restored by supplements of yeast concentrate or marmite to the basal diet of polished rice.
Table I. Effect of Polished Rice and 12 Units Vitamin B1
|Bird No.||Maximum Weight|
|Weight After Depletion on Polished Weight|
|Duration of Depletion|
|Duration of Curative Period|
Figure 1. [Graph showing body weight versus time for pigeons supplemented with vitamin B1 alone and vitamin B1 plus casein. See original for image].
Table II records similar data for those birds on polished rice receiving daily 12 units of vitamin B1 and 2 grams caseinogen.
Table II. Effect of Polished Rice Supplemented by 12 Units Vitamin B1 and 2 Grams Caseinogen
|Bird No.||Maximum Weight|
|Weight After Depletion on Polished Weight|
|Duration of Depletion|
|Duration of Curative Period|
In another experiment two birds, previously brought to maximum weight on mixed corn, were placed on polished rice supplemented with 2 grams caseinogen and 12 units of vitamin B1 daily. After an initial fall of weight of 28 grams and 16 grams, respectively, during the first 4 days, there was almost no change throughout the experimental period, which lasted 37 days (Table III).
Table III. Initial Maximum Weight (Day 0) and Weights After Commencement of Experimental Diet
|Day||Bird 622||Bird 485|
|0||418 g||434 g|
|4||402 g||406 g|
|10||405 g||411 g|
|20||399 g||404 g|
|30||390 g||396 g|
|37||398 g||401 g|
While the attainment of maximum weight on a given diet may, subject to the possibility of storage of essential factors during the initial depletion period, be justifiably held to indicate that such a diet is “adequate” for full nutrition, it does not necessarily follow that it contains a satisfactory balance of essential dietary constituents, such as is present in whole wheat. In two respects a diet of polished rice may be ill-balanced.
In four birds it was found that daily administration of 6 units of vitamin B1 was inadequate with caseinogen, whereas an increase of dosage to 12 units sufficed to promote slow continued rise of weight. This large daily requirement of vitamin B1 may be more apparent than real, since the concentrate in which it is administered is also the source of vitamin B5 [pantothenic acid], another essential dietary constituent for maintenance in the pigeon, and it is possible that the latter may here be a limiting factor.
On the other hand, as suggested by Williams and Eddy (1931), there may be a real difference in the requirement of vitamin B1 for cure of polyneuritis and for maintenance of full nutrition. Morris (1933) claims that, with a basal diet of autoclaved wheat, the dose of a vitamin B1 concentrate necessary to effect complete weight recovery after depletion exceeds the dose required for cure of polyneuritis by an amount that varies from six to sixteen times with different types of yeast concentrate. On the other hand, where the basal diet is one of polished rice, full recovery does not occur even if as much as thirty curative doses of vitamin B1 are administered daily (Carter et al., 1930).
The difference in the nature of the basal diet is important. The factor that appears to limit weight recovery on polished rice supplemented by a yeast concentrate is a relative deficiency of protein. Moreover, a difference in the type of protein available on diets of wheat, on the one hand, and polished rice supplemented by caseinogen, on the other may, perhaps contributes to the difference in the time required for weight recovery in the two cases. Table IV shows the calorie and protein intake for birds on diets of polished rice supplemented by a yeast concentrate, with and without addition of caseinogen.
Table IV. Average Daily Intake [of Test Birds]
|Bird 110||Bird 111|
5 units B1
|Rice + 10 units B1||83.20||1.37||10||60.70||1.00||10||59.27||0.98||10|
|Rice + 12.5 units B1||85.34||1.41||11||55.34||0.92||9||71.42||1.18||10|
|Rice + 2 g caseinogen + 12 units B1||85.0||3.15||52||87.8||3.18||13|
There is, however, in all birds on a rice diet a substantial diminution of the daily protein intake. On the other hand, birds 622 and 401, which rose to their maximum initial weight on a rice diet to which were added 2 grams caseinogen in addition to the yeast concentrate, showed daily calorie and protein intakes that compare favorably with the intakes on whole wheat.
The biological value of the protein of polished rice has been regarded by Osborne et al. (1915) as high, but, on the other hand, its protein content is only about 50 percent of that of whole wheat. There is at present no information as to the minimum protein requirement for the pigeon at maximum weight, and the figures for protein intake on a polished rice diet do not therefore prove that this intake is inadequate. However, the fact that an additional protein supplement to the basal diet, in the form of caseinogen, enables the bird to regain full weight suggests that insufficient polished rice is consumed to meet its protein requirement. In this connection the observations of Aykroyd (1930) on the significance of partial protein deficiency in human beriberi may be mentioned.
It is desirable at this stage to define the sense in which the term “maximum weight” has been used in the preceding experiments. It is not here intended to imply that maximum weight represents some fixed and constant level of nutrition for any particular bird, since the body weight at any given moment is determined not only by the nature of the diet that it is receiving but also by its past dietary history and doubtless by other factors. The birds before experiment are placed on mixed corn for some weeks and during this time come into equilibrium at a weight that has been here termed “maximum.” If, after a period on an experimental diet having polished rice as its basis, the birds are returned to whole wheat, the new weight that is established is not necessarily identical with the former maximum.
In some cases, examples of which will be seen in experiments with wheat germ, the new weight level somewhat exceeds the initial “maximum.” On the other hand, a number of cases have been met with during the course of work in this laboratory in which whole wheat or corn fails to restore the full original weight level. It is not the purpose of this paper to discuss the factors underlying these variations in maximum weight. The facts must be borne in mind in assessing the significance of the experiments with caseinogen that have been described.
Effect of Vitamin B1 and Caseinogen on Pigeon Heart Block
In the experiments described in the preceding section, repeated observations were made on the condition of the heart. At the end of the initial depletion period on polished rice, irregularity of rhythm and bradycardia, which in some cases was very marked, were observed. For example, bird 722, after a depletion period of 24 days, showed a fall of heart rate from 200 [beats] per minute to 66 per minute.
It has been frequently observed that some rise of rate is associated with rise of body weight, though the persistence of irregularity of rhythm shows that the underlying disorder of the vagus centers remains. In all the birds whose weights are recorded in Table II, heart block persisted through the entire period of the experiment, in spite of a weight rise to maximum in four of them. Table V shows records of the heart in two typical birds of this group.
Table V. [Heart Records of Two Birds on Diet of Rice Plus Vitamin B1 and Casein]
|Day of Experiment||Bird 722||Bird 357|
|Heart Rhythm||Heart Rate|
These experiments show that normal nutrition as judged by weight tests can be established in conjunction with a dietary deficiency that is revealed by the presence of cardiac arrhythmia. Experiments now to be described show that this deficiency can be supplied by a constituent of wheat germ.
Effect of Wheat Germ on Pigeon Heart Block
Table VI shows the effect of administration of wheat germ hydrolyzed for 3 hours in association with 12 units of vitamin B1 concentrate.
Table VI. [Effect of Addition of Wheat Germ to Birds on Depleted Diet]
Administration of Wheat Germ
|Daily Amount of Wheat Germ|
|Bird No.||Heart Rate||Heart Rhythm||Weight|
|Heart Rate||Heart Rhythm||Weight|
|622||23||392||140||Dropped beats||376||152||Regular||427||6 g|
|940||39||338||124||“||253||Dropped beats||320||4 g|
It will be seen that in three of these birds that there was a rise in weight that somewhat exceeded the initial weight maximum. The significance of this fact has already been discussed. The remaining birds received wheat germ extract immediately following the depletion period. Birds 921 and 940 received the equivalent of 4 grams of the extract, and in the case of the latter, this dose failed to improve the cardiac condition in a period of 39 days. An increase in the dose to the equivalent of 8 grams resulted in complete recovery of the heart in 4 days.
A final experiment, in which a group of birds were returned to whole wheat after depletion on polished rice, provides further evidence for the view that restoration of heart block can be effected independently of the state of nutrition of the bird as judged by its body weight. Table VII shows that restoration of the heart occurred within 2 to 5 days after commencement of a whole wheat diet, at a time when recovery of body weight was still incomplete.
Table VII. [Restoration of Heart Function Through Whole Wheat Diet]
|Weight After Depletion on Rice|
|Weight at Time of Recovery of Heart|
|Heart Rate After Depletion on Rice||Heart Rate After|
|Time for Recovery of Heart|
|142||488||317||400||100, dropped beats||156,|
|912||346||229||293||128, dropped beats|| 240,|
|23||384||251||310||108, dropped beats|| 184,|
The experiments outlined in this paper provide evidence for the view that one factor limiting the restoration to full weight of pigeons after depletion on a diet of polished rice supplemented by a yeast concentrate is an inadequate allowance of protein in the diet. Where this partial deficiency is made good by the addition of caseinogen, full restoration of weight usually occurs. It is not finally proved that caseinogen, purified in the manner described, provides no factor other than protein, and it would therefore be premature to draw conclusions from these experiments as to the necessity for vitamin B3 in pigeon nutrition. The evidence does, however, indicate that maintenance of or recovery to full weight may be compatible with the persistence of a cardiac arrhythmia of dietary origin. This dietary constituent can be extracted from wheat germ, and its administration in amounts equivalent to 6 to 8 grams leads to complete restoration of function.
I desire to record my sincere thanks to Professor Peters for his continued interest and advice.
By Cyril William Carter, the Department of Biochemistry, Oxford. Reprinted from the Biochemical Journal, Volume 28(3), 1934, by the Lee Foundation for Nutritional Research. Received for initial publication May 5, 1934.
Aykroyd. J. Hygiene, 30:357, 1930.
Carter. Biochem. J., 24:1811, 1930.
Kinnersley and Peters. Biochem. J., 24:1832, 1930.
Kinnersley and Peters. Biochem. J., 21:778, 1927.
O’Brien. Biochem. J., 28:926, 1934.
Osborne, Van Slyke, Leavenworth, and Vinograd. J. Biol. Chem., 22:259, 1915.
Morris. Dissertation, 1933.
Sugiura and Benedict. J. Biol. Chem., 55:33, 1923.
Williams and Eddy. Carnegie Report, 1931.
The Response of Heart Block in the Human to Treatment with Vitamin B Complex
The writer has had a personal experience with “complete heart block” that, after eight weeks, subsided in four days on vitamin B complex [a wheat-germ-rich, whole food supplement created by Dr. Royal Lee]. [After treatment the individual’s] pulse of 28 stepped up to the normal 72, and the character and amplitude of the graph changed from a typical “block” graph to one that was normal.[Image of first heart-response graph, with text:] Before taking vitamin B. [Image of second heart-respond graph, with text:] After four days of treatment. [See end of original document for images of graphs].
—(signed) W.D. [Last name withheld by publisher], MD
Reprint No. 3
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