Biochemical assays of hemoglobin in normal human erythroid clones.

Quantitation of small amounts (2-3 micrograms) of hemoglobin (Hb) was achieved by each of three spectrophotometric techniques. An assay employing benzidine base consistently underestimated Hb at this level and gave a wide scatter of results at higher values. The cyanmethemoglobin method was at the limit of its sensitivity and suffers from the disadvantage of having high optical densities in the blank samples. By contrast, pyridine hemochromogen can be detected accurately with 200 ng of Hb (equivalent of 6,000 mature erythrocytes), and the regression line with this technique virtually passes through the origin. Furthermore, the extinction coefficient of pyridine hemochromogen in the Soret band is 13.5-fold greater than that of cyanmethemoglobin. In normal human erythroid clones, generated in vitro from bone marrow cells, mean cell hemoglobin (MCH) values were determined after various intervals of culture. The MCH after 5, 7, 10, and 14 days were 11.8, 15.8, 26.6, and 34.4 pg, respectively, by the pyridine hemochromogen method. Reaction product was also identified in granulocyte-macrophage clones, presumably reflecting the content of other heme proteins such as catalase and cytochromes. Account must be taken of this non-Hb material in computing true MCH values for erythroid cells.

Anemia due to inadequate iron sources or poor iron utilization.

Anemias due to dietary iron deficiency and poor iron utilization have some features in common. In both, the anemia is hypochromic and microcytic. Also in both, the levels of free erythrocyte protoporphyrin are increased, even though many of the causes of ineffective iron utilization are actually associated with normal or increased iron stores. Appropriate use of currently available assays, including a determination of the level of serum ferritin, can distinguish between many of these disorders. Above all, a logical approach with attention to the clinical response to treatment with iron medications will help achieve a rapid diagnosis.

Control of iron delivery to haemoglobin in erythroid cells.

This paper reviews and reports the results of experiments on the mechanism by which iron is delivered from extracellular transferrin to reticulocyte mitochondria in which haem is synthesized. It is suggested that transferrin donates the iron directly to mitochondria. Transferrin seems to be bound to mitochondria during the process of iron release. When the release of iron from transferrin is blocked by haem, the iron-transferrin complex remains bound to mitochondria so that the total amount of transferrin molecules associated with mitochondria increases in haem-treated reticulocytes. This also leads to an increase in the number of transferrin molecules in the cytosol. In haem-deficient reticulocytes, the rate of dissociation of iron from transferrin is accelerated and the uptake of iron by mitochondria is increased. When the synthesis of haem is inhibited, the non-haem iron in the cytosol (i.e. mainly low-molecular-weight and ferritin iron) comes from mitochondria. Greater amounts of non-haem iron can also be induced in reticulocytes incubated with highly saturated transferrin but, in this case, iron does not seem to be accumulated in mitochondria. These results represent an experimental basis for the elucidation of the excessive non-haem iron accumulation in erythroid cells observed in various clinical conditions.

Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels.

Two heme-peptides (HP) of about 20-A diameter (heme-undecapeptide [H11P], mol wt approximately 1900 and heme-octapeptide [H8P], mol wt approximately 1550), obtained by enzymic hydrolysis of cytochrome c, were sued as probe molecules in muscle capillaries (rat diaphragm). They were localized in situ by a perixidase reaction, enhanced by the addition of imidazole to the incubation medium. Chromatography of plasma samples showed that HPs circulate predominantly as monomers for the duration of the experiments and are bound by aldehyde fixatives to plasma proteins to the extent of approximately 50% (H8P) to approximately 95% (H11P). Both tracers cross the endothelium primarily via plasmalemmal vesicles which become progressively labeled (by reaction product) from the blood front to the tissue front of the endothelium, in three successive resolvable phases. By the end of each phase the extent of labeling reaches greater than 90% of the corresponding vesicle population. Labeled vesicles appear as either isolated units or chains which form patent channels across the endothelium. The patency of these channels was checked by specimen tilting and graphic analysis of their images. No evidence was found for early or preferential marking of the intercellular junctions and spaces by reaction product. It is concluded that the channels are the most likely candidate for structural equivalents of the small pores of the capillary wall since they are continuous, water-filled passages, and are provided with one or more strictures of less than 100 A. Their frequency remains to be established by future work.

Menstrual blood quantitation in the rhesus monkey: an experimental tool for improving intrauterine contraceptive devices (IUDS).

PIP: 6 normally menstruating and 21 ovariectomized rhesus monkeys were used to measure the menstrual blood loss (MBL) during an estrous cycle. A reliable method of quantitating MBL should provide better evaluation of the bleeding problem associated with IUDs. The technic used in measuring the MBL and the volume of MBL are presented. The ovariectomized animals were given 25-mcg estradiol im daily for 22 consecutive days of each month and 1.5-mg progesterone daily during the second half of the injection schedule. These injections supported normal endometrial histology. Intravaginal pads were used for the collection of the menstrual blood. MBL was calculated by the Hallberg-Nillson method. Average MBL for all menstrual periods was 2.01 ml. This should provide a necessary parameter for evaluation of IUD-associated uterine hemorrhage.^ieng