Robert W. Kellermeyer

Iron Overload Disorders: Natural History, Pathogenesis, Diagnosis, and Therapy

Hemochromatosis is a syndrome which, when fully expressed, is manifested by melanoderma , diabetes mellitus, and liver cirrhosis, with iron overload involving parenchymal and reticuloendothelial cells in many organ systems. This clinical presentation may arise as a consequence of either hereditary or acquired abnormalities of iron overload, although the mechanisms are quite different. In hereditary hemochromatosis (also known as primary, or idiopathic, hemochromatosis), increased intestinal iron absorption leads to excessive accumulations of iron, throughout the body, particularly in parenchymal cells. In secondary forms of iron overload including transfusional hemosiderosis, alcoholic cirrhosis, thalassemia, sideroblastic anemia, and porphyria cutanea tarda, iron accumulates in the reticuloendothelial system initially, but with increasing amounts of total body iron, excessive iron deposits eventually accumulate in parenchymal cells throughout the body producing a picture indistinguishable from hereditary hemochromatosis. In this article, the course, prognosis, and therapy of iron overload will be reviewed in detail. Clinical and experimental data concerning the pathogenesis of the different forms of iron overload will be examined critically. In particular, information relating to possible abnormalities of reticuloendothelial function, intestinal mucosal iron transport, and alterations in serum and tissue isoferritin patterns in hereditary hemochromatosis will be analyzed, and possible directions for future research will be suggested. The mode of inheritance and linkage with the major histocompatibility (HLA) complex will be discussed. Theories on the pathogenesis of tissue damage by excess iron will be evaluated. Methods for measuring the extent of iron overload in clinical practice will be described, including measurements of serum iron, serum ferritin, iron absorption, cobalt excretion, desferrioxamine excretion, liver biopsy and tissue iron determinations, and HLA typing. Finally, unresolved problems in the understanding of the disease process, diagnosis, and therapy will be delineated.

Delayed cerebrovascular consequences of therapeutic radiation. A clinicopathologic study of a stroke associated with radiation‐related carotid arteriopathy

A young woman, successfully treated for Hodgkins disease with radiation and MOPP chemotherapy, incurred a devastating stroke months after radiation therapy to the neck and other areas. There was no premonitory clinical history of cerebrovascular attacks. Autopsy showed unilateral thrombotic occlusion of the internal carotid artery unassociated with neoplastic or fibrotic annular constriction of the vessel. There was medial thickening and fibroblastic proliferation within the carotid artery. Areas of focal elastic membrane degeneration involved the cervical portions of the carotid. Thrombus was organized to the damaged vessel wall and was propagated into the intracranial vessels. Aneurysm formation and arterial hemorrhages were absent. These vascular changes occurred in an area of extensive radiation (7200 rads). Pathoanatomical studies in this patient indicate that radiation‐induced vascular changes were associated with a “delayed” stroke.

Thoracic and Elective Brain Irradiation with Concomitant or Delayed Multiagent Chemotherapy in the Treatment of Localized Small Cell Carcinoma of the Lung: A Randomized Prospective Study by the Southeastern Cancer Study Group

A prospective randomized study was carried out to compare the effectiveness of concomitant or delayed multiagent chemotherapy combined with irradiation to the primary tumor and regional lymph nodes and to the brain in a group of 70 patients with histologically proven small cell undifferentiated carcinoma of the lung. Complete and partial response in both groups was comparable, and the overall survival was comparable. However, relapse‐free survival was significantly higher in patients receiving concomitant chemotherapy and irradiation in comparison with the radiotherapy alone group. Disease‐free survival was higher in the concomitant chemotherapy‐radiotherapy patients, although survival was not significantly modified, probably because of suboptimal chemotherapy. The initial intrathoracic failure rate was 40.7% in the concomitant chemotherapy‐irradiation group, compared with 53.8% in the radiotherapy‐alone patients. None of the patients receiving delayed chemotherapy following the radiotherapy recurrence showed signficant tumor response to the drugs. The incidence of distant metastasis was slightly lower in the chemotherapy groups. Brain metastases were noted in 7% of the patients in both groups. Increased intrathoracic recurrences were noted in patients with lower doses of irradiation. Nine of 13 patients treated with inadequate portals developed intrathoracic recurrences in comparison to 13 of 40 treated with adequate irradiation fields. The study emphasizes the need for intensive chemotherapy and adequate radiation therapy to improve survival of patients with small cell undifferentiated carcinoma of the lung. Additional trials are necessary to assess the role of each modality in the management of these patients.

Eosinophilopoietin. A circulating low molecular weight peptide-like substance which stimulates the production of eosinophils in mice.

In earlier studies, methods were developed to raise specific antibodies in rabbits against purified suspensions of mouse or human eosinophils. On administration of antieosinophil serum (AES) to mice, the mature eosinophils in tissues, peripheral blood, and bone marrow were depleted, while the immature eosinophil pool in the bone marrow was observed to proliferate. The current investigations explore the generation of eosinophilopoietic factors during AES-induced eosinophilopenia. Mice received three injections of AES, one every other day. As the peripheral eosinophil counts started to recover after the last AES injection, the serum was collected and transferred to normal animals. Within 2 days the recipients showed an increase in peripheral blood as well as in bone marrow eosinophils. The rise in bone marrow eosinophils was due to newly formed cells as evidenced by increased uptake of [(3)H]thymidine. The generation of eosinophilopoietic activity was specifically related to depletion of eosinophils but not neutrophils. The eosinophilopoietic activity was: (a) dependent on the volume of serum transferred, (b) lost on dialysis, and (c) largely heat labile. The activity eluted as a low molecular weight substance on G-25 Sephadex and was digested by pronase but not by trypsin. Active fractions collected from G-25 columns were not chemotactic for the eosinophils in vitro. Thus, specific depletion of mature eosinophils generates a low molecular weight peptide which stimulates eosinophilopoiesis in vivo. It is suggested that this substance be named eosinophilopoietin.

METABOLISM OF METHYLMALONYL-COA AND THE ROLE OF BIOTIN AND B12 COENZYMES.

The role of methylmalonyl isomerase in metabolism was first elucidated by Flavin et aZ.l in 1955 during their study of propionate metabolism by animal tissue. For orientation we will first consider the role the isomerase reaction plays in this pathway and then its similar role in the formation of propionate by the propionic acid bacteria. Ochoa and coworkers have shown that propionate is metabolized in animal tissue by the following reactions:

Carrageenan and delayed hypersensitivity. II. Activation of Hageman factor by carrageenan and its possible significance.

Carrageenan is a high molecular weight, long-chain polymer of sulfated galactose units that can be extracted from marine plants. Carrageenan has recently been shown to suppress the delayed hypersensitivity reactions of sensitized guinea pigs (1), inhibit the complement system (2, 3), damage macrophages in vitro (4) and in vivo (5), and induce kinin formation in rat plasma (6). In this paper we show that carrageenan activates Hageman factor (factor XII) in human plasma, and thus promotes blood coagulation, induces vascular permeability, and liberates kinin (7). At higher concentrations it also acts as an anticoagulant, as reported previously (8). Materials and Methods. Carrageenan was kindly supplied by Marine Colloids, Inc., Springfield, New Jersey as Seakem 21. The powder was dissolved in hot saline prior to use, using a double boiler technique. Human platelet-deficient plasma was prepared in silicone coated plastic equipment. Venous blood was drawn into 30-ml plastic syringes, mixed with 0.1 vol of 0.13 M sodium citrate pH 5.0 in 40-ml Lusteroid tubes and centrifuged 30 min at 700g at 1°. Plasma was then transferred to thick-walled silicone coated Servall centrifuge tubes and centrifuged at 30,000g for 30 min. Plasma aliquots were then transferred to plastic, silicone coated containers and stored in the frozen state until use. PTA-deficient, Christmas factor-deficient, and Hageman factor-deficient plasma was kindly provided by Dr. Oscar D. Ratnoff. Barbital–saline buffer and DeJalon solution was prepared as described elsewhere (9, 10). Soybean trypsin inhibitor (SBTI) (Worthington) was freshly prepared in barbital–saline buffer at desired concentrations. O-Phenanthroline (Fisher) was prepared in barbital–saline buffer (2.5 × 10–3 M) and stored at 4° for less than 2 weeks. Hexadimethrene bromide (Polybrene, Abbott Laboratories) was similarly prepared and stored. Anti-Hageman factor globulin was prepared as described previously (11) and kindly provided by Dr. Oscar D. Ratnoff.

Production of monospecific rabbit antihuman eosinophil serums and demonstration of a blocking phenomenon.

Abstract Eosinophilia is the hallmark of helminthic, allergic and some neoplastic conditions, but the role of the eosinophil remains largely unknown. To elucidate eosinophilic function specific antiserums were prepared with methods developed to produce monospecific antimouse eosinophil serum. Purified suspensions of eosinophils from a patient with Hodgkins disease and another with leukemia when injected into rabbits resulted in antieosinophil serums containing high titers of agglutinating and cytotoxic antibodies to eosinophils. Both antiserums were monospecific for eosinophils, having no cross-reactivity with neutrophils. Serum from the eosinophil donor with Hodgkins disease contained immunoglobulins that were cytotoxic to eosinophils and blocked the effect of the antiserum prepared from his own cells. Monospecific antihuman eosinophil serum may be useful to study pathologic states associated with eosinophilia. (N Engl J Med 290: 417–420, 1974)

[35] 2-methylmalonyl-CoA mutase from Propionibacterium shermanii (methylmalonyl-CoA isomerase)☆

Publisher Summary This chapter discusses the 2-Methylmalonyl-CoA Mutase from Propionibacterium shermanii in depth. The bacteria are grown, harvested, and extracted. Methylmalonyl-CoA-2- 14 C is converted by enzymatic isomerization to succinyl-CoA-3- 14 C. The reaction involves not transcarboxylation, rather a movement of the carboxylthioester group from the 2 to the 3 position of the propionic acid moiety. These results do not indicate whether the transfer occurred intermolecularly or intramolecularly. In the mutase reaction, it is likely that the B 12 coenzyme accepts hydrogen from a carbon adjacent to the methyl carbon of methyl-malonyl-CoA thus, permitting a partial bonding of the CoA group to the adjacent or the number 2 carbon. Using mass spectrometric techniques and 18 O-labeled substrates, it was demonstrated that an oxygen atom is transferred from C-2 to C-1 in the conversion of propane-l, 2-diol to propionaldehyde. Although no metal requirement is noted for methylmalonyl-CoA mutase, the dioldehydrase, another B 12 dependent enzyme, is stimulated by K + and NH 4+ ions. Reaction rates with the purified mutase reported here showed no stimulation of K + ions and NH 4 + ions up to a final concentration of 0.33 M.

Mechanical Fragility of Erythrocytes from Patients with Paroxysmal Hemoglobinuria.

Summary 1. As pH of blood samples from 6 normal subjects was decreased, susceptibility of normal erythrocytes to mechanical trauma or their mechanical fragility was increased. 2. MF determinations on PNH and normal erythrocytes were done at various values of pH including the range for optimum complement activity in normal serum and it was found that mechanical trauma did not contribute to hemolysis of normal or PNH erythrocytes even though the PNH hemolytic mechanism was active. 3. It is postulated that increased red cell volume due to a shift of C1-, HCO3- and H20 into the erythrocyte, may be the mchanism of increasing spheroidicity of the erythrocyte and the MF as a function of decreasing values of pH. Conclusion. 1. MF of erythrocytes of the PNH patient in an active properdin system does not differ from MF of the same erythrocytes in an inactive properdin-complement system, nor does the MF of these erythrocytes differ from that of normal erythrocytes at values of pH between 8.0 and 5.9. 2. The hemolytic system of PNH, which requires properdin, complement and Mg+ +, does not enhance susceptibility of PNH erythrocyte to hemolysis by mechanical trauma in vitro. Hemolysis of PNH and normal erythrocytes attributable to mechanical trauma increases progressively as pH of blood decreases until a critical point (pH 6.0-6.2) where MF rises rapidlv.

[33] Methylmalonyl-CoA racemase from Propionibacterium shermanii☆

Publisher Summary This chapter describes the enzyme that is prepared from Propionibacterium shermanii (52W). The (S)-methyhnalonyl-CoA formed as a result of racemase action is converted to propionyl-CoA and oxaloacetate through the oxaloacetate transcarboxylase reaction. The oxaloacetate is then converted to malate with malate dehydrogenase, and the velocity of the reaction is determined from the decrease in absorbancy of the NADH at 340 mμ. Methylmalonyl-CoA racemase is unusually resistant to heat. Compared to an unheated control, 67% of the activity remained after exposure to boiling water for 1 minute and 50% remained after 5 minutes. This property could also be used to advantage in the purification procedure. The epimerization does not involve transfer of the coenzyme moiety between the two carboxyl groups. It apparently attacks and destroys the asymmetry of carbon 2. The proton can then enter from either side, permitting racemization.