The presence of large numbers of lipid droplets in the cytoplasm of most cortical cells is known as a feature of the adrenal gland of the dog. In the present study histological sections were stained with various selective stains and compared with those stained with sudan black. Lipid staining was performed by the SCHULTZ test, SMITH-DIETRICH reaction, and CIACCIO method. Twelve young dogs of both sexes were employed. Frozen sections of the unstimulated gland contained a large number of sudanophilic lipid droplets of varying size. Inanition of a dog for seven days caused a decrease in sudanophilic lipid droplets of the zonae fasciculata and reticularis. Daily injections of cortisone acetate to starved dogs inhibited the depletion of sudanophilia in the inner zones of the cortex.
The intensities of the SCHULTZ test and the SMITH-DIETRICH reaction paralleled the intensity of the sudan stain in every case of the present experiment. The total lipid content determined by the sudan stain was considerably higher than that determined by any of the selective stains.
The distribution of lipid droplets demonstrated by the method of CIACCIO was quite different from that of sudanophilia in frozen sections. In normal dogs, CIACCIO-positive lipid (CPL) droplets were presented only in the zona glomerulosa and found in some of the cortical cells bordering a sinusoid. In starved dogs, CPL droplets increased markedly in number and size. Repeated injections of cortisone acetate caused an accumulation of CPL in the zona fasciculata.
The amount of CPL in the zona glomerulosa was almost the same as that in the controls.
Changes in adrenal lipid were already described by many workers in the rat adrenal cortex given various stimuli. The lipid changes clarified in the present experiment were similar to those observed by previous workers in frozen sections. However, the results of investigation on CPL were peculiar. The CIACCIO method has scarcely been used in the study of adrenal lipid probably due to the general disrepute in which this method has been held. The author is of opinion that is reliable if its technique is carried out strictly.
Such peculier changes of CPL as observed in this experiment have not yet been noted.
It has been known that the adrenal cortex is characteristically a lipid-poor structure in the hamster and ruminants. It was suggested that the distribution and alteration of lipid in the adrenal cortex were not the same in these animals as in the other animals. The distribution and histochemistry of CPL coincided exactly with those of the lipid which had been reported to be present in lipid-poor adrenals. Accordingly, the CPL detected in this experiment might probably be the same with solitary lipid in lipid-poor adrenals.

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Experiments were performed to know whether some essential amino acids were deficient in grass protein and whether the addition of such dificient amino acids to grass protein was able to increase the biological value of proteins for rats.
The concentrated parts of red clover (RC) and Kentuchy 31 fescue (KF) was prepared by the structural analysis method described in the previous paper (1959). The concentrated part contained 46.7 and 27.8 per cent crude protein on the basis of air-dried matter in RC and KF, respectively.
In this experiment, amino-acid analysis was first made chemically by applying column chromatography. Then deficient amino acids were searched for indirectly by measuring twice the biological value of protein.
The method of MOORE and STEIN (1951) was employed for amino-acid analysis. It was found that RC and KF were almost the same in the amino-acid composition of the concentrated part, which was as follows: serine 4.24 (4.21), glutamic acid 12.99 (11.15), glycine 5.70 (6.08), alanine 6.24 (6.75), valine 6.50 (6.75), methionine 1.10 (1.19), isoleucine 5.78 (5.34), leucine 10.94 (9.04), tyrosine 4.19 (3.51), phenylalanine 7.06 (6.44), tryptophan 1.49 (1.31), histidine 2.47 (2.44), arginine 6.15 (5.86), and lysine 6.34 (5.53) in RC and in KF (shown in parentheses) when expressed in grams per 16g of nitrogen. In this composition, aspartic acid and threonine were determined together as a single substance in the protein of RC, because they could not be separated in this analysis. As a result, glutamic acid and the peak which contained aspartic acid and threonine were found to be the highest and leucine was the second highest. Methionine was the lowest amino-acid content. Trytophan, histidine, and tyrosine were found to be the second lowest contents.
To measure the biological value of protein for rats, a diet containing a 10% level of protein was used. The results of determination of the amino-acid contents of the concentrated part in RC and KF were compared with the amino-acid requirements for young rats presented by ROSE (1937). It was proved that four amino acids, methionine, lysine histidine, and tryptophan (with threonine only in the case of KF), were more or less, deficient in these diets. Then trials were carrid out to examine the following five different diets for biological value and digestibility: (1) diet containing the concentrated part (CP) of RC as the sole protein source, (2) that containing the CP of KF as the sole protein source, (3) that containing the CP of RC supplemented with such amino acids as DL-methionine, L-lysine-HCl, and Lhistidine-HCl-H₂O, (4) that containing the CP of KF supplemented with such amino acids as mentioned above and DL-threonine, and (5) that containing casein supplemented with DL-methionine and L-lysine-HCl. Diet No. 5 served as control.
The results obtained showed that neither RC protein nor KF protein had any high biological value, which was as low as 29 and 44, respectively. When essential amino acids were supplemented to RC and KF proteins, the biological value of each grass was increased remarkably, reaching 97 per cent in RC and 94 per cent in KF. This fact indicates that the grass proteins were deficient in the amino acids added to the diets. The digestibility of grass protein was also found to increase to some extent when the same deficient amino acids were added to the diet. The rate of increase, however, was not so high in the digestibility as in the biological value. Some difference was seen in the increase of digestibility between RC protein and KF protein.

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The proteins of orchard grass (Dactylis glomerata) at various growth stages were examined for digestibility by the two different pepsin-digestion methods described below. Then the cause of the difference in digestibility between the two methods was discussed.
The methods were carried out as follows.
"Digestion A": A grass sample was ground by a blendor and extracted successively with water, 0.3% NaOH, and hot-0.3%-alkaline-60%-ethanol. Protein nitrogen "a" was determined in each extracted fraction (and the insoluble fraction). On the other hand, the grass ground wasd igested by pepsin at 37°C for 48 hours in citrate-HCI buffer (pH 1.2), extracted by solvents, and determined for protein nitrogen "b" in each fraction. Then the digestibility of each Protein was calculated from the formula a-b/a×100.
"Digestion B": An aliquot of the extracted fraction was used to determine the protein nitrogen content "c" of the fraction. Another aliquot was incubated with pepsin at 37°C for 48 hours, the incubation mixture having been adjusted to pH 1. 2, and used to determine residual protein nitrogen "d". Digestibility was then calculated from the formula c-d/c×100.
Results obtained are as follows.
1. The digestibility of water-soluble protein was generally much lower in "digestion B" than in "digestion A". In NaOH-soluble protein, digestibility was generally much higher in "digestion B" than in "digestion A". In hot-alkaline-ethanol-soluble protein, digestibility was higher in "digestion A" than in "digestion B". In insoluble protein, digestibility was much lower in "digestion B" than in "digestion A".
2. The sequence of digestibility in "digestion B" among proteins in the grass was as follows: water-soluble>NaOH-soluble>hot-alkaline-ethanol-soluble>insoluble. The sequence of digestibility in "digestion A" was as follows: water-soluble>hot-alkaline-ethanol-soluble>insolubl>NaOH-soluble.
3. The above-mentioned results seem to, suggest that not only digestion of proteins but also some change in solubility of the proteins took place during the process of "digestion A". That is, it seems that water-soluble and hot-alkaline-ethanol-soluble proteins lost their solubility in each solvent, and that a part of the NaOH-soluble protein in the digested sample was derived from some other protein(s).
4. The digestibility of the whole protein (the sum of the extracted proteins and insoluble protein) calculated in "digestion B" was considerably lower than that calculated in "digestion A:. This result suggests that there was a change in property of the proteins during the extraction.
5. Digestibility in "digestion B" decreased in NaOH-soluble and hot- alkaline-ethanol-soluble protein and whole protein as grass grew in the first growth stage. This shows a possibility that a change occurred in the quality of these proteins during the growth of grass.

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Two drying processes, wilting for 7 days in the laboratory and heating at 70°C with ventilation, were examined for effect on the digestibility of proteins in orchard grass (Dactylis glomerata).
The proteins in the grass were fractionated into 5 groups, water-, 0.3%-NaOH-, and hot-alkaline-60%-ethanol-soluble and insoluble protein, using successive extractions with these solvents.
The two digestion methods described in the previous paper were applied. In "digestion A", grass samples were ground and digested with pepsin before various forms of protein nitrogen were determined. In this case, digestibility was calculated comparing the value of protein N in each fraction before and after pepsin-digestion. In "digestion B", the fractions extracted with the solvents (and the insoluble fraction) were treated with pepsin. Digestibility was determined from a decrease in protein nitrogen.
The results obtained are as follows:
1. Although the amount of total nitrogen in the grass changed neither by wilting nor by heat-drying, the protein nitrogen (the sum of the protein N in each fraction) in the fress grass decreased to a large extent by wilting, but slightly by heat-drying (table 1).
Conspicuous changes in amount of proteins in each fraction by drying were a decrease in water-soluble protein N and an increase in water-soluble non-protein N by wilting and a decrease in water-soluble protein N and an increase in hot-alkaline-ethanol-soluble protein N by heat-drying (table 2).
2. In wilted grass, digestibility "A" was higher than digestibility "B" in water-soluble, hot-alkaline-ethanol-soluble, and insoluble protein, showing the same trend as in fresh grass.
3. Water-soluble protein showed a little change in digestibility "A" by wilting. On the other hand, the digestibity "A" of hot-alkaline-ethanol-soluble and insoluble proteins decreased remarkably by wilting.
4. After wilting, digestibility "B" decreased considerably in water-soluble and hot-alkaline-ethanol-soluble proteins, scarcely changed in NaOH-soluble protein and decreased remarkably in insoluble protein.
5. By heat-drying, digestibility "B" decreased a little in water-soluble protein, increased considerably in NaOH-soluble protein, hardly changed in hot-alkaline-ethanol-soluble protein, and decreased remarkably in insoluble protein.
6. The digestibility of protein as a whole was a little lower in wilted grass than in fresh grass by digestion "A", much lower in wilted and in heat-dried samples than in fresh grass by digestion "B".

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Histological changes were observed in the hypothalamo-hypophysial system and the anterior hypophysis of the rabbit during pregnancy, parturition, and lactation, from the viewpoint of neurosecretion.
The results obtained are as follows.
1. During pregnancy, a comparatively large number of cells with enlarged nuclei and vacuoles were observed in both supraoptic and paraventricular nuclei. On the 25th day of pregnancy, reaccumulation of densely-packed or colloid-like granules was seen in the peripheral zone of neurosecretory (NS) cells. Granules was seen in the hyphothalamo-hypophysial tract (HHT) and the neurohypophysis increased in number on the 5th, 15th, and 20th days of pregnancy and decreased slightly on the 10th and 25th days, suggesting a cyclic fluctuation in the NS activity of the hypothalamus during pregnancy. Granules were transferred increasingly from within the HHT and the pars nervosa into the pars anterior on the 5th, 15th, and 20th days of pregnancy. The number of granules transferred decreased slightly on the 10th and 25th days, but it was still larger than that in the normal control. On the 5th, 15th, and 20th days of pregnancy, an increase of acidophils and degranulation of both acidophils and basophils were observed in the pars anterior.
2. At parturition, a large number of cells with enlarged nuclei and cytoplasmic vacuoles were observed in both supraotic and paraventricular nuclei. These cells had less granules. Granules decreased markedly in number in the pars nervosa. Degranulation was visible particularly in the peripheral zones of blood vessels, suggesting a release of the oxytocic principle into the blood stream. NS granules increased in number in the pars intermedia and the pars anterior. Similar stainable substance was seen in the pituitary cleft. Degranulation was observed in both acidophils and basophils of the pars anterior. Cytoplasmic vacuolization was strikingly apparent in the basophils.
3. During the period of lactation, pictures of both promoted granule-elaboration and vacuole-formation were observed in NS cells with functional nuclear enlargement. In the pars nervosa, degranulation was frequently observed in the peripheral zones of blood vessels, especially 5 minutes after suckling stimulus was given by young. In the lactating period, a comparatively large number of NS granules were seen in the pars intermedia and the pars anterior. Such granules increased markedly in number especially 5 minutes after suckling stimulus was given. A great amount of the same stainable substance was present in the pituitary cleft. In the animal exhibiting the pictures mentioned above, NS granules were clearly seen in the ventral part of the pars anterior. In the pars anterior, degranulation of both acidophils and basophils was usually observed, especially 5 minutes after suckling stimulus was given. Peripheral vacuoles were seen in a large number of bashphils. From the morphological evidence of lactating animals, it seems highly probable that tactile stimuli given to the mammary gland pass the afferent nerve pathways to activate the NS mechanism in the hypothalamus, which in turn excites the secretion of the oxytocic principle from the pars nervosa, and that this NS substance penetrates the pars anterior from within the HHT and the pars nervosa through the hypophysial portal vessels and the pars intermedia, and promotes the release of lactogenic hormone to the pars anterior.
From the results mentioned above, the penetration of NS granules into the adenohypophysis coincided, in time and lacation, with the increase of acidophils and the appearance of degranulation of basophils. Therefore, it was postulated that the stainable NS substance might be the neurohumor stimulating the release of anterior-pituitary hormone.

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The casein complex was studied for a relationship between the sedimentability and the contents of some constituents by measuring the nitrogen, calcium, and various forms of phosphorus of the supernatant liquids of unheated or heated (85°C, 30 minutes) skim milk resultingfrom ultracentrifugation for serially increasing time. The effects of cooling and adding acid and calcium salt were also studied. The results are asfollcws.
1. About 11 per cent of the casein was not sedimented even after ultra-centrifugation at 35, 000G for 60 minutes. The precipitated nitrogen of heated milk was more than that of unheated milk. This increased precipitate in heated milk, at least a part of it, was assumed to be composed of denatured whey protein contained in the casein complex.
2. The calcium: nitrogen ratio and the phosphorus (in various forms): nitrogen ratio were essentially the same among different sizes of the casein complex. Besides, these ratios were not affected by heating.
3. The nitrogen precipitated by 60 minutes' centrifugation was decreased when the pH was lowered from 6.7 to 5.6-5.2 by addition of lactic acid. It was increased again at the isoelectric point of casein (pH4.6). Calcium and inorganic phophorus were released from the casein complex by addition of acid.
4. By addition of calcium chloride, the precipitable nitrogen was increased. At the same time, the calcium: nitrogen and the inorganic phosphorus: nitrogen ratios of the precipitate were increased, so that the calcium: inorganic phosphorus ratio remained almost constant.
5. The precipitable casein was decreased by cooling, which did not markedly affect the calcium: nitrogen and the inorganic phosphorus: nitrogen ratios of it.

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The method for extraction of gonadotropins from the urine of goat was investigated. Specimens of human urine collected from postmenopausal and normally menstruating women were used for a preliminary study. Twenty-four-hour urine samples were collected from cyclic and pregnant goats of the Japanese Saanen breed. Crude urinary gonadotropic substances (crude UGS) were extracted from these urine samples by means of a modification by the authors of the kaoline-absorption method developed by Bradbury, Brown and Brown. Biological assays of crude UGS were conducted with the enlargement of the uterus in injected immature mice as criterion. When the uterine weight was increased by at least 100% of that of the control animal, it was considered that a positive response was exhibited. A mouse uterine unit (MUU) was defined as the smallest amount of crude UGS which made 50% of the injected mice exhibit a positive response.
The kaolin method used by the present authors is as follows.
1. Add 5 volume percent of 20% kaolin suspension to acidified-filtrated urine (pH4.5) and stir thoroughly for 5 minutes.
2. Discard the supernatant and collect the precipitated kaolin fraction. Add accidified water (pH4.5) and stir vigorously. Decant the supernatant and wash the precipitate repeatedly with pH-4.5 water so that the kaolin may be free from urine.
3. Add 1N ammonia water and stir thoroughly for 20 minutes. After centrifugation, add ammonia water again to the precipitate, stir, and centrifuge. Combine the second ammonia eluate with the first.
4. Adjust the combined eluates to pH8.5 with 2N acetic acid and centrifuge. Adjust the supernatant to pH5.5 and centrifuge. Add 5 volumes of cold acetone to the supernatant and keep it in the refrigerator overnight. Collect and dry the precipitate.
Gonadotropic activity could be determined in crude UGS derived from human urine by means of biological assay, but could not in those derived from goat urine due to the toxic effect of contamination found in them. No positive response was given by mice injected with crude UGS derived from goat urine and purified by some simple procedures.
It was concluded that the kaolin-extract from goat urine had a toxic effect which would affect the general condition of animals used for assay and thereby influenced the responsiveness of the end-point in the biological assay. Therefore, it was required to purify further crude UGS derived from goat urine.

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In the previous report, it was mentioned that crude UGS (gonodotropic preparations extracted from the urine by the kaolin method) derived from the goat had a severe toxic effect on animals used for assay, and that it was impossible to determine the activity of partially purified preparations from crude UGS derived from goat urine by biological assay.
In the present experiment, kaolin extracts from the urine of the goat were purified further by means of the modification by the authors of the liquid-chromatography method developed by Butt and Crooke. The absorbent used was composed of 10ml of 8% suspension of tricalcium phosphate and 1.5g of hyflo-super-cel. The amount of crude UGS prepared from 100ml of original urine was dissolved in 1ml of 0.85% sodium chloride solution and poured on the column. The material was absorbed by the top of the column. The elution of this material was conducted with two eluting agents. The first fraction which had been eluted from the column by 20ml of 0.002 M disodium hydrogen phosphate was designated the GA fraction and the second fraction eluted by 10ml of 0.02 M trisodium phosphate, the GB fraction.
The sugar contents of the GA and GB fractions were measured by the use of orcinal reaction. In this reaction, 1ml of the GA or GB fraction was added to 1ml of a solution of 1.6% (w/v) orcinol in 30% (v/v) sulfuric acid, and then 3ml of 80% (v/v) sulfuric acid was added. The combined solution was placed in a water-bath at 80°C for 30 minutes and then immediately cooled at 4°C for 15 minutes. The color density of this solution was read in a spectrophotometer at 420mμ. Glucose was used as the standard.
The gonadotropic activity of the GA plus GB fractions was assayed biologically with the increase of the mouse uterine weight as criterion. The GA plus GB fractions were mixed slowly with 10 volumes of cold ethanol. The precipitate was named (GA+GB) EtOH ppt. and stored in a desiccator until it was subjected to biological assay.
When (GA+GB) EtOH ppt. prepared from the urine of cyclic and pregnant goats was assayed biologically for gonadotropic activity, positive responses were elicited. One mouse uterine unit (MUU) of the preparation was produced from 300 to 400ml (about 8-hour equivalent) of original urine. It was considered that the value of sugar measured by chemical assay was related closely to the biological activity of the preparation.
It was concluded that the procedure consisting of kaolin extraction and tricalcium-phosphate purification was useful for detection and determination of the gonadotrodie activity in the urine of the goat.

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For the purpose of getting a more insight of the mechanism of the discoloration and the browning of butter fats during the oxidation, changes in the spectral characteristics occurring butter fats in the course of autoxidation and oxidation by heating were studied.
Fresh butter fats had the absorption maxima at 230, 270, 280, 302, and 317mμ in the ultraviolet absorption spectra and at 430, 455, and 482mμ in the visible region of the spectra. It was known that the absorption maxima in the visible and the ultraviolet regions of the spectra were produced by carotene pigments and sterols, respectively.
The absorption maxima of butter fats in the visible region disappeared prior to the begining of oxidation of the fats. The absorption maxima in the ultraviolet region disappeared gradually in the course of oxidation of the fats.
Conjugated diene and triene acids were contained about 1 and 0.02 per cent, respectively, in fresh butter fats. There was no difference in the content between these conjugated acids in butter fats in the course of autoxidation.
Autoxidized and colored butter fats and the oxidized-acid-free fractions of butter fats were studied for spectral characteristic. It was recognized that the coloration of fats resulted from the increase of absorption of light in a wide range from the ultraviolet to the visible regions in the absorption spectra of the fats.

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