Catharine S. Rose

Bifidus factor. IV. Preparations obtained from human milk

Abstract 1. 1. Cream, proteins, and salts can be removed from human milk with only small losses of bifidus activity. 2. 2. For the separation from lactose, processes have been developed using adsorption and elution on charcoal or fractional precipitation. 3. 3. The lactose-free, active concentrates upon hydrolysis gave acetic acid and four sugars, d -glucosamine, l -fucose, d -glucose, and d -galactose. All of the latter were identified through crystallized derivatives. Acetylation gave inactive chloroform-soluble, peracetyl compounds from which the bifidus-active N-containing oligo- and polysaccharides could be regenerated. 4. 4. By chromatography on charcoal and on paper, at least four different active components have been found. All of them contained N -acetyl-glucosamine units. All were levorotatory but one, which was lacking in l -fucose and was dextrorotatory. 5. 5. The nitrogen-free saccharides obtained from the lactose-free concentrates of human milk by chromatography did not show any bifidus activity.

Bifidus factor. II. Its occurrence in milk from different species and in other natural products

Abstract The activity of human milk for the growth of Lactobacillus bifidus var. Penn was compared with that of other species. The average relative activity was highest for human colostrum, closely followed by rat colostrum, then by human milk, rat milk, and cows colostrum. The milk of ruminants (cow, ewe, goat) showed only very slight activity. Somewhat higher activity was found in the milk of cat, monkey, dog, donkey, rabbit, mare, and sow. The bifidus growth factor has been demonstrated in high concentration in various secretions in man, such as saliva, semen, amniotic fluid, meconium, and tears. Hog gastric mucin which has a high blood-group titer has also shown a high titer for bifidus factor, while other nitrogencontaining polysaccharides were free or almost free of bifidus factor. Ammonium salts and N -acetylglucosamine as well as N -acetylgalactosamine, especially the ammonium salts, in high concentration, were active, probably as precursors of the growth factor.

Availability of vitamin E in the newborn infant.

Summary Red blood cells of newborn infants are hemolyzed by dilute solutions of hydrogen peroxide. This hemolysis may be prevented by vit. E in vitro or in vivo. However, even with high daily doses of vit. E (500 mg of mixed tocopherols) given the mother during the last weeks of pregnancy the hemolysis test in the newborn remained unaltered.

Preparation of alkyl N-acetyl-α- and -β-d-glucosaminides and their microbiological activity for Lactobacillus bifidus var. Penn☆

Abstract The preparation of the lower alkyl N -acetyl-α- and -β- d -glucosaminides has been described using N -acetyl- d -glucosamine and the corresponding alcohol in the presence of a cation exchanger. Chromatographic separation resulted in the isolation of the crystalline methyl, ethyl, and n -propyl N -acetyl-α- d -glucosaminides and crystalline methyl, ethyl, and n -propyl N -acetyl-β- d -glucosaminides. The alkyl N -acetyl-β- d -glucosaminides were found to be active as growth factors for Lactobacillus bifidus var. Penn whereas the corresponding alkyl N -acetyl-α- d -glucosaminides were inactive. The ethyl and n -propyl N -acetyl-β- d -glucosaminides were considerably more active than the methyl compound.

TOCOPHEROL AND HEMOLYSIS IN VIVO AND IN VITRO

Work of Houssay and Martinez,l published in 1947, suggested that the same mechanisms might be active in protecting the rat against the toxic effects of alloxan that had been found of value in protection of the liver against acute massive necrosis induced by dietary means. Among other effects of dietary factors, they observed longer survival after alloxan injection in rats which had received methionine (but not choline) as a supplement to a diet high in lard, or had been given a diet in which the lard was replaced by vegetable oils. I t has recently been shown that tocopherol is a protective agent for the liver, and experiments were undertaken to determine whether the advantage of vegetable fats might depend on their content of tocopherol. The rapid hemolysis of the blood of tocopherol-deficient rats, which was the major finding of this investigation, was observed first at autopsyS2 The kidneys of rats which died a few hours after injection with 160 mg. per kg. of alloxan intraperitoneally were completely engorged with blood. Hemoglobinuria and hemoglobinemia were then followed, and the extent of hemolysis in some cases was astounding, Within 10 minutes after the injection, the centrifuged hematocrit tube showed the serum layer dark red in color, and, in about half an hour, the layer of cells, instead of being about 50 per cent of the total volume, was almost undetectable. In those animals which survived, the hemoglobin was excreted and there was no further hemolysis. TABLE 1 shows the effect of variations in the tocopherol-deficient diet

Prolonged feeding studies in premature infants

Summary Two hundred seventy-seven premature infants were fed formulas of varying composition in an attempt to evaluate growth (length and weight) and metabolic response. (The composition of the 5 formulas used is given in Table I.) Premature infants grew well for prolonged periods when fed formulas which provided 3.0 to 8.0 Gm. protein per kilogram per day. Infants fed formulas which provided more or less protein did not grow as well. Human milk formula alone did not support comparable growth of premature infants. Results obtained from feeding a formula of cows milk with a protein content of 1.1 per cent were identical with those in infants who were fed human milk. Infants who were fed formulas which provided over 5 Gm. protein per kilogram per day had persistently elevated blood urea nitrogen levels and excreted large quantities of urinary phenols. Other biochemical or growth differences in premature infants who were fed the other specified formulas were small. There were no statistical differences in the serum protein values in infants who were fed 3 to 8 Gm. of protein per kilogram per day. In contrast, infants who were fed less protein have shown lowered serum protein values. The food efficiency ratio was identical for formulas with 1.5 to 4.1 Gm. of protein and lower in those with less. In contrast, the values for the protein efficiency ratio indicate better utilization of protein for the formulas with low-protein content than for those with higher-protein content. The lowest value was found for the formula with 4.1 per cent protein.

Microbiological Studies on Growth Factor for L. bifidus var. pennsylvanicus

Summary 1. The results of microbiological assays on a large number of oligosaccharides obtained from human milk and on simple derivatives (disaccharides or simple synthetic compounds) of N-acetyl-D-glucosamine are reported. The high microbiological activity of 4-0-β-D-galactopyranosyl-N-acetyl-D-glucosamine and of ethyl- and n-propyl-N-acetyl-β-D-glucosamine has not been exceeded by any other compound tested. 2. The “true” specific bifidus factor has either still to be found or several related compounds may act as precursors or in synergistic combination.

Bifidus factor. III. The rate of dialysis

Abstract 1. 1. The rates of dialysis of Lactobacillus bifidus factors have been measured under standard conditions for various samples of human milk and human colostrum. For comparison glucose, lactose, and melezitose have also been used. The latter dialyze more rapidly than the bifidusactive factors. 2. 2. The blood group of the donor (A, B, O) seems to have little effect on the rate at which the activity dialyzes. 3. 3. The initial activity of the samples decreases with the length of time after delivery. However, the distribution after dialysis for 72 hr. remains relatively constant. 4. 4. For human colostrum, the dialysis is slower and less complete than for human milk collected 3–49 weeks after delivery.

Tocopherol requirements of rats by means of the hemolysis test.

Summary The sensitivity of the red blood cells of tocopherol-deficient rats to the hemolyzing action of dialuric acid in vitro has been applied to the study of tocopherol requirements. Rats which had received the stock diet until they were 2 to 3 months old showed signs of deficiency after only a few days on a tocopherol-deficient diet. Animals which had received large supplements of tocopherol were protected many months after it was discontinued. About 3 mg of DL-α-tocopherol per day per kg of body weight was required for protection of the erythrocytes. The newborn young of rats which had received the stock ration were susceptible to the hemolyzing action of dialuric acid. If the females were given a supplement of tocopherol throughout the gestation period the blood cells of the young rats were protected. The possible application of this hemolysis method to the bioassay of vitamin E has been discussed.

Bifidus factor. V. The activity of α- and β-methyl-N-acetyl-d-glucosaminides

Abstract 1. 1. β-Methyl- N -acetyl- d -glucosaminide is a growth factor for Lactobacillus bifidus var. Penn . The corresponding α-glucoside is inactive. 2. 2. When mixtures of the inactive α-methyl- N -acetyl- d -glucosaminide and of the active β-isomer are used, the activity of the β-isomer is enhanced up to fivefold. 3. 3. Other compounds containing NH-CO groups did not enhance the activity of β-methyl- N -acetyl- d -glucosaminide. In general, no or only very slight enhancement by the α-glucoside was observed when the β-glucoside was replaced by other substances with bifidus factor activity. 4. 4. Lactobacillus bifidus var. Penn contains an enzyme that hydrolyzes and inactivates β-methyl- N -acetyl- d -glucosaminide but not the corresponding α-glucoside. In the presence of the α-glucoside the rate of hydrolysis of the β-glucoside was decreased. The observed in vitro effect did not seem sufficient to explain the microbiological response.