The results of this study may be stated as follows.
1. Rapid injection of more than 0.06 of a gram per kilo of hemoglobin intravenously into a normal animal is followed by the appearance of hemoglobin in the urine (pelvis of kidney) within eight to ten minutes.
2. After rapid injection of more than 0.012 of a gram per kilo per minute of hemoglobin, 16 to 36 per cent. of the total amount, if this equals 0.25 of a gram per kilo, is eliminated in the urine and is accompanied by choluria.
3. If the injection of not more than 0.35 of a gram per kilo is made slowly (less than 0.01 of a gram per kilo per minute), the amount eliminated in the urine is only 2.33 to 9.5 per cent. of the total amount injected, and choluria does not occur.
4. The concentration of free hemoglobin in the blood which constitutes the threshold value of the kidneys for hemoglobin is approximately 0.06 of a gram of hemoglobin per kilo of body weight. When about this concentration is reached, hemoglobin appears in the urine.
5. The amount of hemoglobin per kilo of body weight which, after rapid injection, may be retained without jaundice, is approximately 0.18 of a gram. When 0.22 or 0.23 of a gram is retained bile pigments appear in the urine. The threshold of the liver for jaundice in point of hemoglobin saturation lies, therefore, between 0.18 and 0.22 of a gram per kilo of body weight. With slow injections a greater amount may be retained without choluria.
6. The absence of the spleen does not alter greatly the percentage of hemoglobin eliminated by the kidney, nor does it raise the threshold of the liver for jaundice.
7. In the presence of jaundice, either hemolytic or obstructive, the amount of hemoglobin retained by splenectomized animals is slightly diminished and that eliminated by the kidneys is correspondingly increased.
Upon these data may be based the following explanation of the mechanism by which free hemoglobin is removed from the blood serum. Hemoglobin is not removed by the kidney until its concentration in the blood serum reaches a certain level (0.06 of a gram of free hemoglobin per kilo of body weight). This constitutes the threshold value of the kidneys for hemoglobin and when it is reached hemoglobin appears in the urine. When the concentration is lower, hemoglobinuria ceases; at the same time, however, the liver, and possibly other tissues, take up hemoglobin as soon as mere traces are present in the serum and they continue this removal whether the renal threshold is exceeded or not. The two processes go on simultaneously, the rate of removal, when the renal threshold is exceeded, being for the kidneys 17 to 36 per cent., and for the liver and other tissues 64 to 83 per cent, of the total amount introduced. The hemoglobin which is removed by the liver is transformed into bile pigments. If the amount reaching the liver is small and is received slowly, the amount of bile formed is not increased above the excretory capacity of the liver, and it is removed by the bile passages without the occurrence of choluria. This is shown in our experiments in which injections of hemoglobin were made more slowly than 0.01 of a gram per kilo per minute. On the other hand, if the hemoglobin is taken up by the liver rapidly and in large amounts, the bile capillaries are overtaxed and the bile cannot be rapidly removed, but is reabsorbed into the blood, and choluria develops.
If this theory is correct we have an explanation of those instances of blood destruction in man characterized by jaundice, but not accompanied by hemoglobinuria. In a slow, gradual destruction of the red blood cells, the liver removes the hemoglobin from the serum so rapidly that the concentration of hemoglobin in the serum does not reach the threshold value of the kidneys and hemoglobinuria, therefore, cannot occur. The constant absorption of large amounts of hemoglobin by the liver and the increase in bile formation which results does, however, overtax the bile passages and jaundice occurs.
In the same way may be explained the continuance of jaundice after the disappearance of a transient hemoglobinuria. A rapid destruction of a large amount of blood raises the concentration of hemoglobin in the serum so quickly that the threshold value of the kidney is quickly exceeded and hemoglobin appears in large amounts in the urine. When an amount of hemoglobin sufficient to reduce the concentration of the serum below the threshold value of the kidney has been removed, a considerable amount of hemoglobin may still remain in the serum, and it is the slow elimination of this through the liver that causes the choluria to continue.
The demonstration that the absence of the spleen has no important influence on the elimination of hemoglobin by the kidney, on its transformation into bile pigments, or on the removal of such pigments, is of interest in connection with an observation made in the first paper of this series. This was concerning the frequent failure of jaundice to follow the administration of hemolytic serum during the early period following splenectomy. Among the possible explanations was the suggestion that the spleen is in some way concerned in the disintegration of free hemoglobin or in the elaboration of its derivatives. The present investigations demonstrate that such an explanation is without experimental basis, though it does not controvert the possibility of the spleen being concerned in liberating hemoglobin from the red cells and suggests that the failure of jaundice is due to some other factor or factors. Evidence to indicate that the changes in the blood that follow splenectomy are important factors is offered in the third paper of this series.