M. C. Kik

Hematopoietic Function in Avitaminosis. VI. Vitamin G Deficiency.

In a study of the secondary anemia of childhood, Happ and Evans 1 pointed out that 9 of their 10 patients had rickets, although the anemia could not be attributed to a lack of vitamin D, since it did not improve following the addition of cod liver oil. Since in human cases we seldom encounter uncomplicated avitaminosis, for the reason that diets that are deficient in one or 2 vitamins are generally deficient in other respects, particularly in minerals, we have found it of interest to determine if an anemia could be produced on the Steenbock-Black ration No. 2965 2 which is now extensively used by nutritional workers in vitamin research for the production of uncomplicated rickets in the rat. For this study we have employed 39 animals, 30 on the Steen-bock and Black ricketic diet No. 2965 and 9 controls receiving the same diet supplemented with vitamin D by irradiating the ration 30 minutes with a mercury quartz vapor lamp. Bleedings of most of the rats were performed twice weekly and of the others once weekly. Daily records were kept of body weight and food intake. At each bleeding hemoglobin and specific gravity determinations and erythrocyte counts were made. In Table I we present the hematopoietic function of a representative animal receiving the Steenbock and Black ration 2965 supplemented with vitamin D, showing anemia, as evidenced by the concentration of hemoglobin and erythrocyte counts. This is the typical picture that was obtained on the ricketic animals as judged by the line tests, 3 and the same blood picture was encountered on all the 9 control animals that showed normal calcification. We, therefore conclude that vitamin D deficiency has no influence on hematopoietic function.

Influence of the Maternal Diet on Concentration of Hemoglobin of Nursing Young of Albino Rat.

In a recent communication 1 we have reported on “Blood Formation of the Albino Rat on a Standard Dietary Regime”, of the following composition: whole wheat, 25; rolled oats, 26; yellow corn, 25; oil meal, 15; commercial casein, 5; cod liver oil, 1; CaCO3, 0.5; NaCl, 0.5; and a liberal supply of whole milk daily. On that ration, designated as stock diet 1, we found the concentration of hemoglobin of nursing baby rats appreciably below that reported by Williamson and Ets. 2 We are now finding considerably higher values on a different type of diet, designated as ration 1145, of the following composition: casein (purified) 20; dehydrated bakers yeast (Northwestern), 10; McCollums salt mixture No. 185, 4; butter fat, 5; and dextrin, 61. Our findings are submitted graphically in Chart I. All the lactating mothers were raised on stock diet 1. During the reproduction period the females were divided into 3 groups. One group was mated and allowed to rear the young on stock diet 1; another group was transferred during the later part of pregnancy to ration 1145; and the third group was transferred to ration 1145 on the date of the birth of young. The curve of hemoglobin concentration of nursing young on maternal stock diet 1 represents 416 determinations. 1 For our work on maternal diet 1145 we made 432 determinations, 18 young having been taken for each age. Since we found no significant increase in the concentration of hemoglobin of the nursing young, whose mothers received ration 1145 during the latter part of gestation compared with that of the nurslings whose mothers received the same diet on the date of birth of the litters, we are showing only the figures of the latter group.

Avitaminosis. XIV. Effect of Vitamin A Deficiency on Concentration of Blood Lipids of Albino Rat

Although it is well known that fats act as carriers of certain vitamins, nothing definitely has been established on the influence of avitaminosis on lipid metabolism. The literature is conflicting, because the difficulties involved in the analytical chemical methods are many. Another reason for inconsistent data, obtained on adequate control diets, is probably due to the fluctuating results produced by metabolic changes incident to the normal processes of digestion and absorption of lipids in the animal organism. In our lipid metabolism work we have fasted our animals one hour previous to bleeding. In order, however, to eliminate all possibilities of the influence of food on blood lipids, we are continuing our studies, fasting the rats for much longer periods. In the meantime, we feel that a summary of our results completed would be of interest to the clinician as well as to the nutritional investigator. The dietary technique employed has been described in a previous communication. 1 The details of our blood methods will be submitted in a subsequent communication elsewhere. The blood determinations were made once weekly and the experimental period lasted 50 to 65 days. For this study a total of 64 rats was employed, 27 controls and 37 pathological animals. In Tables I and II are submitted typical illustrations representative of the remainder of the group. The pathological states of the animals during the advanced stages of the avitaminosis were associated with loss of weight, severe xerophthalmia and pneumonia, but as shown in Tables I and II the concentration of the blood fatty acids, cholesterol, and phospholipids fall within the same range as that of the controls, which made excellent growth without any apparent symptoms of vitamin A deficiency.

Hematopoietic Function in Avitaminosis. IV. Further Studies of Vitamin A Deficiency.

In a previous publication 1 we have suggested the occurrence of a secondary anemia in vitamin A deficiency. Our results, however, were not conclusive. We, therefore, continued this investigation with 30 more animals, 18 of which received our vitamin A deficient ration 1749.† Twelve of these rats were allowed a modification of this diet, so that one to 2% of lard was replaced by equivalent amounts of butterfat, in order to prevent sudden deaths from pneumonia frequently associated with this avitaminosis. In addition, we have studied the effect of vitamin A deficiency on the differential leucocyte count in 9 females and 9 males. In the females the disease was followed with daily examinations of the vaginal smears according to the technique of Evans and Bishop. 3 Daily records were kept of food consumption, and in the majority of animals records were also kept of the daily water intake. Bleedings were performed on each animal twice weekly and determinations were made of hemoglobin and erythrocyte counts. Specific gravity determinations were also made, in order to secure information on blood concentration. The experimental period ranged from 68 to 173 days. To summarize our results, we found no noteworthy disturbance in hematopoietic function during various stages of this avitaminosis, as characterized by the severity of ophthalmia and loss of body weight, nor any changes in the differential leucocyte count during the onset, and remission produced by vitamin therapy. Our results, therefore, do not substantiate the findings of Koessler and coworkers. 4 Although secondary anemias have been encountered in keratomalacia in man, 5 the investigators admit that other complications or infections associated with this disease may be the influencing factors rather than this avitaminosis.

Effect of methionine on casein metabolism.

Summary Methionine promotes the nitrogen retention of casein and in this experiment, proved even better than cystine.

Avitaminosis. IX. Influence of Vitamin A Deficiency on Albumin-globulin Ratio of the Blood of Albino Rat.

Since it is now generally recognized that infection accompanies vitamin A deficiency, 1 and since it is claimed that during infection there is a change in the albumin-globulin ratio of the blood, 2 due to the rise of the globulins, it was thought of interest to investigate if in this avitaminosis there is produced a shift in the albumin-globulin ratio of the blood of the albino rat. A total of 18 animals were used in this study, 12 pathological and 6 controls. The micro methods used for the determinations of albumin and globulin were those perfected by Greenberg. 3 Weekly determinations were made for periods ranging from 63 to 102 days. The animals were 49 to 61 days old when started on the experiment and weighed 82 to 127 gm. Summarizing all of our results for the entire experimental periods, the pathological animals showed an albumin-globulin ratio of 2.0 and the controls a ratio of 1.7. Only 4 animals out of the 12 pathological showed any considerable fall in the albumin-globulin ratio during the terminal stages of the avitaminosis, as indicated by a ratio of 1.3 in 2 animals and a ratio of 1.5 in 2 others. From the character of our results we conclude that the albumin-globulin ratio cannot be used as an index of infection in vitamin A deficiency.

Avitaminosis. VIII. Effect of Vitamin B Deficiency on Concentration of Non-protein Nitrogen of Blood.∗:

Since during advanced stages of vitamin B deficiency there is considerable tissue catabolism, it was thought of interest to investigate if in this avitaminosis there are any significant changes in the non-protein constituents of the blood of the albino rat. The total non-protein nitrogen of the blood was studied in 8 pairs of rats on diets deficient in the vitamin B complex and in 2 pairs on rations deficient only in vitamin B. Weekly determinations were made. Litters were restricted to the same plane of nutrition as the pathological animals but in addition received either the vitamin B complex or vitamin B in the form of dehydrated Northwestern bakers yeast. In addition, 3 sets of animals were studied in groups of 4. The first one of each group received the vitamin B deficient diet (vitamin G having been supplied by autoclaved N. W. yeast); a litter mate received vitamin B but was restricted to the same amount of food and water as the pathological animal; a third litter mate received the same ration as the second, but water ad libitum; and the fourth litter mate received the same diet as the second, and food and water ad libitum. In these animals the concentration of the non-protein nitrogen, urea, and uric acid of the blood were determined once weekly. The age of the animals was 50 to 68 days. The weight was from 65 to 91 gm. and the experimental period lasted from 14 to 155 days. Summarizing the results on the vitamin B complex, there was an average concentration of 39.7 mg. non-protein nitrogen per cent in the pathological animals and 37.7 mg. per cent in the controls receiving vitamin B, but on the same plane of nutrition as the avitaminotic rats.

Vitamin Requirements of Nursing Young. VIII. Effect of Vitamin B Deficiency on Alkaline Reserve of Blood of Nursing Young of Albino Rat.

In order to determine the influence of uncomplicated vitamin B deficiency on the alkaline reserve of the blood of nursing young of the albino rat, 84 nursing young, whose mothers received stock diet No. 6† were sacrificed during the later part of the lactation period, and the carbon dioxide volume per cent determined on blood secured from the carotid artery. We used the manometric method of Van Slyke and Neill 2 on samples of 0.1 cc. of plasma. Since in the pathological nursling there has been, because of stunting of growth, a considerable prolongation of the nursing period, 6 additional animals on maternal stock diet No. 6, 32 days of age, were taken during the post-lactation period. The range of carbon dioxide volume per cent found was 37 to 65. The 72 pathological animals examined were in a condition of prolonged maintenance, or in a condition of prolonged maintenance accompanied by incipient polyneuritis. The avitaminosis of the nurslings was produced on maternal diet 1438, 3 adequate in every respect with the exception of vitamin B. Only 4 pathological animals showed a deviation from the normal which occurred during the last stages of polyneuritis. These results are in agreement with the findings of Sure and Smith 4 on growing and adult rats.

Enzymic Digestion of Lactalbumin Versus Casein in Vitro.

Since the experimental evidence concerning the nutritive value of lactalbumin is conflicting, an intensive study has been initiated in this laboratory to investigate the biological value of this protein from the standpoint of digestion, metabolism, and growth. Casein (being a protein of indisputable excellent biological value) was used as a standard of comparison. In this report a summary is given of the results of tryptic-ereptic digestion of lactalbumin versus casein in vitro. The casein was furnished by Merck and prepared according to the method of Hammersten. The lactalbumin was obtained from Harris Laboratories which is the purest product obtainable with a minimal ash content. The technique employed has been described. 1 The proteins were studied in 3 concentrations, 120, 180, and 210 mg. As a source of trypsin and erepsin pancreatic and intestinal extracts were taken from rats of our colony stock diet, on which ration excellent growth is obtained.

Enzymatic Efficiency in Malignancy.

Recently we have begun a detailed investigation of enzymatic efficiency of avitaminosis. 1 Being at the same time engaged in a problem on the relationship of vitamin therapy to regression of tumors in the albino rat, we had access to considerable numbers of tumor-bearing animals and had available the necessary materials and equipment to study the enzymatic efficiency of malignancy. We found our Wistar stock of animals rather resistant to the Flexner-Jobling carcinoma; consequently, the greater portion of our work was done on animals receiving another type of transplantable tumor, in which we had from 60 to 85% takes, provided the tumor used for implantation was not too necrotic. The tumor used was furnished by Dr. Francis Carter Wood of New York City, which was designated as Walker Carcino-Sarcoma No. 256. The experiments were conducted in pairs, and for every animal with an implanted tumor, a litter-mate control of the same sex was used, which was restricted to the same plane of nutrition as the tumor-bearing animal. The tumors were measured with a caliper in length and width, and in some cases in depth. Forty-two groups were studied in the case of most enzymes. The blood serum phosphatase was included later in the investigation and was studied in 26 groups. The tumors ranged from 15 to 73 mm. in length and from 10 to 65 mm. in width. The age of the tumors of the implanted animals, the blood and tissue enzymes of which were studied, ranged from 3 to 5 weeks in most cases, but some individuals were allowed to proceed to the stage of pronounced ulceration and these tumors were about 7 weeks old at the time the animals were sacrificed. The tumors were embedded in formalin and examined microscopically by Drs. Thatcher and De Groat in Little Rock.