Esther M. Greisheimer

Individual variation in serum calcium in normal men and women.

Since the work of Bell 1 many investigators have studied the blood calcium level in relation to menstruation. The results of these investigations have been contradictory. Watchorn 2 , Close and Osman 3 , Allen and Goldthorpe 4 , Spiegler 5 , and others have concluded that there is very little or no change in the blood calcium in relation to the menstrual cycle. Sharlit et al 6 and Matters 7 report a premenstrual rise in blood calcium and a menstrual fall in the calcium level. Okey et al 8 state that “while the changes in the concentration of serum calcium at any phase of the monthly cycle are not outstanding, there is some tendency to frequency of low values for calcium a few days previous to the onset of menstruation and to frequency of higher values from the 8th to the 15th days following the onset of menstrual bleeding.” With the exception of the work of Okey et al 8 , these findings have been based upon either the study of very small groups of individuals or upon blood calcium determinations taken not more than 3 or 4 times during a menstrual cycle. Experimental work done on lower animals shows that the blood calcium level is definitely affected by the reproductive system. Riddle and Reinhart 9 found a tremendous increase in calcium in the blood of birds prior to ovulation. Mirvish and Bosman 10 have produced a decrease of 2 to 3 mg. in the plasma calcium in both rabbits and human beings by injecting ovarian follicular and corpus luteum extracts. The present study was undertaken in an effort to determine standards of blood calcium in relation to the menstrual cycle in women with normal menstrual periods in order that we might have some basis for comparison in a study being made on a group of women with dysmenorrhea.

Serum Calcium in Relation to Menstruation in Cases with Dysmenorrhea.

A study of the serum calcium in relation to the menstrual cycle in a group of 10 women who had dysmenorrhea was made. The cases selected were those having severe dysmenorrhea necessitating bed rest every month. Our purpose was to determine whether women having dysmenorrhea showed any significant variations in calcium level from women with normal menstrual periods. Serum calcium determinations were made in duplicate on 10 dysmenorrhea cases twice a week for 4 consecutive weeks. The blood was drawn between 8 and 10 a. m. The determinations of serum calcium were made by one of us (E. M. G.) using the Clark-Collip 1 modification of the Kramer-Tisdall 2 method. A total of 80 readings were made. Table I presents the range in the serum calcium and the mean of the 8 determinations for each individual in the group. The mean low reading for the group was 10.23 mg., the mean high reading 11.01 mg., giving a mean range of 0.78 mg. In a group of women with normal menstrual periods studied by us, 3 the range was 1.15 mg. This difference in range, however, is not sufficient to be of significance. The mean reading for the entire group was 10.57 mg. In our group of normal women we found a mean of 10.01 mg., again a difference of no significance. Table II presents the mean serum calcium for each week of the menstrual cycle. The lowest reading of 10.49 mg. occurs in the rest period, while in the normal group, the lowest reading occurred in the menstrual week. The apparent cyclic variation in calcium levels in relation to menstruation found in the women with normal menses, with a pre-menstrual rise and post-menstrual fall in serum calcium was not observed in this group with dysmenorrhea.

Glycogen Formation in Rats. III. Diets Containing 87.5 Per Cent of Total Caloric Value in Lactose, Glucose and Sucrose.

Each diet contained 87.5 gm. of the carbohydrate, 14.7 gm. of casein (85% casein) and 4 gm. of salt mixture 185. This gave a caloric value of 3.77 cal. per gm., of which 87.5% was derived from carbohydrate and 12.5% from protein (not considering digestibility). Each diet was supplemented by vitamin feeding daily. 1 Adult rats which had served for breeding purposes until discarded, were used. The time on the test diet varied from 16 to 18 days. The food and water intake was measured. The technique followed at the end of the test period was similar to that previously described. 2 The rats fed on the lactose diet developed a diarrhea. They disliked the diet as evidenced by their scratching it from the food box, consequently we could not measure the intake accurately. As nearly as we could tell, the average daily intake was 7.9 gm., and the intake during the last 24 hours was about the same. The daily intake on the glucose diet averaged 13.3 gm. with 14.4 gm. as the last 24-hour intake. The sucrose daily intake averaged 14.3 gm., with 14.0 as the average for the last 24 hours. The water intake on the lactose diet averaged 27 cc. per day, influenced, of course, by the diarrhea. On the glucose diet it was 13.9 cc., and on the sucrose diet 10.8 cc. The mean body weights are given for the beginning and end of the test period: Lactose (6 females, 4 males) 283.9 gm.—246.3—loss 37.6 gm. Glucose (6 females, 4 males) 311.6 gm.—314.2—gain 2.6 gm. Sucrose (3 females, 2 males) 307.8 gm.—318.7—gain 10.9 gm. The liver glycogen averaged 3.15 ± 0.12% on the lactose diet; 4.89 ± 0.21 on the glucose, and 4.89 ± b 0.20 on the sucrose.

Influence of Diet on Action of the Sulfonamide Drugs.

The importance of diet in the normal physiology of the liver and in the protection of the liver from the action of such damaging substances as chloroform and carbon tetrachloride has been brought to the attention by many investigators. 1-7 Since we found that sodium sulfapyridine exerts a definite and characteristic effect on the levels of blood sugar and liver glycogen, 8 we wished to study the effects of various diets on the action of sodium sulfapyridine and sodium sulfathiazole in rats. The diets used to date are Purina Dog Chow Checkers as the balanced diet, and experimental diets in which protein, carbohydrate and fat form about 87% of the caloric value of the diet. 9 In these studies the rats were kept on the special diets for one week, and were not fasted before the experiment. The daily food intake and gain or loss in body weight during the last week were recorded. This information, together with the blood sugar, liver glycogen (expressed both as per cent of liver weight and as mg per 100 g of body weight), free, total and acetylated levels of the drug in the blood is shown in Table 1. One dose of the drug to be studied was given by intraperitoneal injection. 7.5% solutions were used; 1 cc per 100 g of body weight was given. The animals were killed 3 hr later. It will be noticed that the administration of sodium sulfapyridine to rats that have been on the balanced diet leads to a decided decrease in liver weight, an increase in blood sugar and a decrease of 68% in liver glycogen (calculated from mg of liver glycogen per 100 g of body weight). The changes which follow the administration of sodium sulfathiazole are in the same direction but less marked; the level of the drug in the blood was lower in this group.

Fasting Blood Sugar in Rats

It was found that the blood sugar during fasting falls more rapidly in women than in men. 1 The study has been continued on rats. The animals were divided into 3 groups; the first group was fasted 12 hours, the second, 24 hours, and the third, 42 hours. Water was allowed during the fast. At the close of the fasting period, the rats were decapitated, and the blood sugar determined in duplicate by the Shaffer-Somogyi method. The values found are shown in Table I. The total fall for the female rats was 27.8%; of this 67% occurred between 12 and 24 hours; the total fall for the male rats was 20.9%; of this, 59% occurred between 12 and 24 hours. These results agree with those found in men and women in that the blood sugar falls more rapidly in females during fasting.

Sexual differences in fasting blood sugar.

Investigators have disagreed as to the changes in blood sugar during fasting; those who have reported a decrease have failed to note a sexual difference. Allen believed that the blood sugar is maintained at 100 mg.% through prolonged starvation, almost up to death. Joslin 2 found the blood sugar decreased from 120 to 90 mg.% during 4 days, in which a healthy woman fasted, save for small quantities of weak bouillon. Weeks and his co-workers 3 observed 64 epileptics, who fasted 3 weeks. Sugar determinations were made only once a week; the sugar was reported as being maintained at an approximately even level. Acidosis was present, but not severe. Shaw and Moriarty 4 observed epileptic children during 10 to 14 day fasts. They found a steady diminution in blood sugar, with the minimum (46 mg.%) between 3 and 8 days, after which the sugar increased. They considered the diminution in blood sugar peculiar to childhood, since it was supposed that the blood sugar is not much diminished in adults during fasting. Lennox and his co-workers 5 observed 16 men and 8 women during 3 to 21 day fasts. They noted that the blood sugar fell during the first week of fasting. Their minimum was 65 mg.%. Shope 6 studied one woman who fasted 6 days. He found that the blood sugar decreased to a minimum of 37 mg.%. As the fast continued, a secondary rise in blood sugar occurred. Deuel and Gulick 7 observed 5 men and 5 women during 7 day fasts. The women subjects showed ketosis before the men. It developed more rapidly and became more severe, as shown by both the urine analyses and the carbon dioxide combining power of the plasma. No blood sugar studies were made.

High Carbohydrate and High Fat Diets

Summary The glucose tolerance was not significantly altered by a very high carbohydrate intake, over a period of 7 days. After 7 days on a high fat diet, the response to the glucose tolerance test was typical of diabetes mellitus; the tolerance fapidly returned to the usual type, after return to the balanced diet. Since the diet can so markedly influence the tolerance, this should be kept in mind in glucose tolerance studies. The urea nitrogen and total non-protein nitrogen vary markedly with different dietary conditions. This fact is of clinical importance. The fasting blood sugar is markedly influenced by a diet low in carbohydrate. A high intake of carbohydrate seemed without effect on the fasting blood sugar. It was planned to make more extensive studies, but the subject developed undulant fever, and the experimental work was necessarily postponed.

Glycogen and water storage.

MacKay and Bergman 1 stated that in rabbits there is a tendency for water to be stored in direct proportion to glycogen storage in the liver. The same authors 2 found that in young albino rats, on the average 3.8 gm. of water are stored with each gram of glycogen. That there is a relationship between glycogen and water storage is denied by Puckett, 3 Holmquist, 4 and Bridge. 5 In view of the difference of opinion noted above, the authors decided to study the question. Adult rats (in most cases) of the Long-Evans strain were used. All the animals were kept on the McCollum Stock Diet I (whole wheat ground, 67.5%, casein 15%, whole milk powder 10%, butter fat 5.2%, calcium carbonate 1.5%, and sodium chloride 0.8%). The rats were killed by decapitation; the blood sugar was studied by the Shaffer-Somogyi method, the glycogen in a portion of the liver by the method described previously, 6 and the water content in the remaining portion of the liver by drying in an oven to constant weight at 105°C. In the first study 48 adult and 6 young rats were used. They were not fasted, but allowed the stock diet ad libitum until the time of killing. The results for the males and females are combined, and shown in Table I. The relationship between glycogen and water content was tested by the determination of the correlation coefficient. When the percentage of glycogen and the percentage of water are used in the calculations, the correlation coefficient is +.202 ± .088, which would indicate a lack of correlation. However, when the absolute values, as glycogen per liver and water per liver, are used in the calculations, the correlation coefficient is +.745 ± .041.

Glycogen Formation in Rats. IV. Diet Containing 87.5 Per Cent of Total Caloric Value in Maltose.

Fifteen healthy young male rats, varying from 145 to 226 gm. in weight, were given us by Dr. Palmer of the University Farm. They had been on a normal diet, and had not been subjected to experimental procedure. We placed 5 of them on McCollums Stock Diet I, for controls. The remaining 10 were placed on a diet containing 87.5 gm. of maltose, 14.7 gm. casein (85% casein), and 4 gm. of salt mixture 185. This diet had a caloric value of 3.77 per gm., of which 87.5% was derived from maltose and 12.5% from casein, not considering digestibility. The analysis and caloric value of the stock diet was given in a previous publication. 1 The Maltose diet was supplemented by vitamin feeding daily. 2 The experiment was in progress for 16 days. Both the control and the experimental rats gained in weight. The former averaged 3.4 gm. per day, the latter 3.2 gm. The average daily intake of food was 14.13 gm. on the stock, and 14.19 gm. on the maltose diet. The daily water intake averaged 20.76 cc. on the stock, and 15.91 cc. on the maltose diet. The following results were found: The difference in liver weight is not statistically significant. The liver glycogen formed on the maltose diet is significantly higher than that formed on the stock diet. If we consider the last 24-hour intake in terms of calories per gram of body weight, we find 0.2785 for the stock, and 0.2854 for the maltose diet. If the liver glycogen, expressed in terms of milligrams per gram of liver, be divided by the caloric intake, the quotient is 175 for the stock and 208 for the maltose rats.

Relationship Between Fibrin Content and Sedimentation Index in Normal Individuals.

The series of investigations was carried out on normal individuals between 18 and 30 years of age. It includes 52 women and 50 men. The sedimentation index was determined by the Cutler method and the fibrin by the Wu colorimetric method, with slight modifications. The fibrin determinations were done in duplicate. The average sedimentation index for women was 7.635 (mm.) ±.345; the average index for men was 2.380 ± .164. The difference between the 2 sexes is 5.255 ± .382. This difference is 16.3 times as large as its probable error, and is probably significant. The average fibrin content for women was .300 (gm. per 100 cc. plasma) ± .004. For men it was .288 ± .004. The difference between the 2 sexes is .012±.006. Since this difference is only twice as large as its probable error, it cannot be considered certainly significant. Treatment of the sedimentation indices and the fibrin contents by the statistical method gave a correlation coefficient of .312±.084 for the women. This is 3.83 times as large as its probable error and is probably significant. The empirical mean fibrin values as ordinates are plotted against the sedimentation indices in the graph. While certain of these means, particularly that for the fibrin content of .325 (index 1), deviate rather widely from the regression line, it must be remembered that the total number of cases is 52, and that certain of these means are based on very few analyses. The correlation coefficient for men was .328±.085, which is 3.86 times as large as its probable error, and is probably significant. The empirical mean fibrin values plotted against the sedimentation indices, and the fitted regression line is shown in the graph. The difference between the correlation coefficients for the 2 sexes is only .016±.120.