Experimental Studies on Catabolism of Hemoglobin On the Metabolism of Double-Labeled Hemoglobin-Haptoglobin Complex.

Hemoglobin haptoglobin complex injected intravenously into rats is removed primarily by the liver and subsequently is digested. The use of a single isotope, however, to label and to pursue the Hb-Hp complex in many of the previous reports has led to some confusion concerning the metabolic fate of the complex. To elucidate the mechanism of degradation of Hb-Hp complex to a higher degree of accurary, a doubly labeled Hb-Hp complex was prepared from Hb labeled with 59Fe in the heme moiety and from purified rat haptoglobin tagged with 125I. This was injected intravenously into rats which were then sacrified at timed intervals. Prior to the injection, uncomplexed Hb or non-Hp impurities were removed from the tagged complex by gel filtration on a Sephadex G-100 column. The purity and stability of each batch of labeled complex were confirmed prior to injection. All specimens were measured for radioactivity in a well type NaI scintillation detector coupled with a standard spectrometer. Dual isotope counting was performed using the two window method. In normal rats, blood radioactivities after injection of the complex in closes of 0.4 mg as Hb per head of rat decreased rapidly at an exponential rate. However, there was a marked difference between the plasma clearance of TCA (trichloracetic acid) precipitable 59Fe and 125I activities during the early phase, 5 to 30 minutes after the injection. It was noted that the latter was faster than the former. Likewise, an appearance of free 125I derived from the catabolized Hp moiety was observed as early as 5 minutes after the injection. And the appearance of 59Fe in the TCA supernatant in the form of transferrin iron was delayed by almost 60 minutes as compared with the appearance of the former. Discrepancies were also noted in the tissue distribution of the two nuclides. During a three hour observation, 125I activity in the liver remained at a relatively constant value of 18% of the injected close whereas 59Fe activity increased steadily reaching 42% of the close at 180 minutes after the injection. Induction of acute inflammation by turpentine oil or inoculation of Yoshida sarcoma into rats resulted in acceleration of the clearance of the injected Hb-Hp complex. Tissue distribution studies disclosed that the rate of organ uptake of the complex was also accelerated in these two groups of rats. The rate of 59Fe accumulation in the liver was slightly faster in tumor bearing animals when compared with those of inflamed rats. In contrast, subsequent uptake of radioiron by the bone marrow of the inflamed rats was definitely higher than normal or tumor bearing rats. It is concluded, therefore, that the Hb-Hp complex given via an intravenous route is generally removed by the liver and bone marrow, the degradation of the complex takes place. Not only are the rates of Hb and Hp catabolism influenced by the presence of a tumor or inflammation, but the rate of reutilization of iron liberated during the course of heme breakdown will also be influenced by the presence of similar pathologic conditions.