Lactic acidosis as a result of iron deficiency.

Iron-deficient rats have an impaired work performance, even when their anemia is corrected by exchange transfusion. Muscle activity is associated with a higher blood lactate concentration than is observed in iron-replete animals. The accumulation of lactate is a result of excessive production as lactate clearance from the blood was shown to be unaffected. By adjusting the work load to a lower level, it was possible to divide iron-deficient animals into two groups, one capable of continued treadmill running and another in which animals stopped before 20 min. In the former, blood lactate concentration reached a plateau at moderate levels, whereas it continued to increase in the latter until the animal stopped running. Levels of alpha-glycerophosphate oxidase in skeletal muscle mitochondria were found to be much lower in the second group (P < 0.001). Lactate infusion into normal animals was shown to interfere with work performance, and maintenance of a normal pH in iron-deficient and iron-replete animals did not prevent the impairment in work associated with high blood lactate concentrations. Additional evidence was obtained that energy substrate (blood glucose and free fatty acids, muscle glycogen) was adequate in irondeficient animals. Oxygen tension in their vena caval blood was higher than in controls. Furthermore, the in situ behavior of electrically stimulated gastroenemius and soleus muscles appeared similar to that of control animals. Because the stimulation of the single muscle in the iron-deficient animal did not result in appreciable elevation of blood lactate and did not show impaired contractility further supported the hypothesis that the elevation of blood lactate caused the decreased work performance. It is concluded that iron deficiency by a depletion in the iron-containing mitochondrial enzyme, alpha-glycerophosphate oxidase, impairs glycolysis, resulting in excess lactate formation, which at high levels leads to cessation of physical activity.

Iron deficiency in the rat. Physiological and biochemical studies of muscle dysfunction.

Work performance on a treadmill has been evaluated in normal and iron-deficient rats. Anemia was removed as a variable by adjusting the hemoglobin of all animals to the same concentration. At a hemoglobin compatible with normal work performance, iron-deficient animals showed a marked impairment of running ability as compared to control animals. Iron therapy corrected the disability within 4 days. Concentrations of the cytochrome pigments and myoglobin, and rates of oxidative phosphorylation with pyruvate-malate, succinate, and alpha-glycerophosphate as substrates were all reduced in mitochondrial preparations from skeletal muscle of iron-deficient rats, but only the rate of phosphorylation with alpha-glycerophosphate as substrate increased significantly and in parallel with the recovery in work performance of the iron-deficient rats treated with iron dextran.

Cytotoxicity responses to melanoma cells by human lymphoid cell subpopulations.

Lymphoid cell subpopulations from normal donors and patients with malignant melanoma were assessed for cytotoxicity. Unfractionated mononuclear cells and T-cells from melanoma patients gave cell-mediated cytotoxic (CMC) responses to melanoma target cells but not to human fibroblasts. These specific CMC responses to melanoma cells were partially inhibited by autologous serum. Non-T-cells and nonrosetting cells from melanoma patients were not directly cytolytic for melanoma target cells; however these same subpopulations were cytotoxic in the presence of autologous serum, which indicated antibody-dependent cell cytotoxic responses. Non-T-cell subpopulations from normal donors were not cytotoxic when incubated with autologous serum. A third cytotoxic mechanism was demonstrated with complement (C3) receptor-activated lymphocytes. From both melanoma patients and normal donors, cells forming rosettes with erythrocyte-antibody-complement were nonspecifically cytotoxic for lung fibroblasts and melanoma target cells, These responses were independent of antibody, since melanoma serum presumably containing antibody to melanoma target cells neither enhanced nor blocked cytotoxicity mediated by C3 receptor-bearing lymphocytes. The results indicated that lymphoid cell subpopulations from the same melanoma patient could express at least three different lymphocyte-mediated cytotoxicity mechanisms against melanoma target cells.

Electron transport systems of Candida utilis: purification and properties of the respiratory chain-linked external NADH dehydrogenase.

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 x 10(6). The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ6 and CoQ1 derivatives as acceptors. Rotenone (10(-5) M) and seconal (10(-3) M) do not inhibit enzymatic activity.

Cytochrome a3 deficiency in human achondroplasia.

Mitochondria prepared from tissue culture cells (skin fibroblasts) from normal subjects and subjects with homozygous achondroplasia were studied to determine the concentrations of cytochromes a and a3 in the preparations. Cytochrome a3 was markedly decreased (80%) in the achondroplastic preparations with cytochrome a present in normal amounts. Determination of total heme a (as the pyridine hemochromogen) in the normal and achondroplastic preparations demonstrated that the observed decrease in concentration of cytochrome a3 in the achondroplastic preparations was due to an absence of cytochrome a3 and not to a change in its absorbancy (extinction coefficient). The decreased concentrations of cytochrome a3 in the achondroplastic cells may decrease the reactivity or affinity of the mitochondrial oxidative systems for oxygen and result in the phenotypic expression of the disease.

A longitudinal study of T and B lymphocytes from a three-year-old patient with severe combined immunodeficiency (SCID) in 'gnotobiotic protection'.

Fluctuations in the percentages and absolute numbers of T and B lymphocytes were observed in the peripheral blood of a patient with severe combined immunodeficiency maintained in a gnotobiotic environment. Up to 24 months of age, 72-86% of the lymphocytes had surface membrane immunoglobulin (SMIg), 37-47% bore a receptor for C3(EAC-RFC), and 3-12.5% formed spontaneous rosettes with sheep erythrocytes (E-RFC). These values persisted until 30 months, after which shifts in the percentages and absolute numbers of T and B cells were observed. A significant decrease in the proportion of SMIg-bearing cells to 20-40% (169-405 mm3), and EAC-RFC to 10.5-39% (114-259 mm3), was accompanied by a general increase in the proportion of T cells to 19-60% (141-1026 mm3), representing a lymphoid subpopulation approach to normal levels.

Reactive oxygen species and DNA oxidation in fetal rat tissues.

It is well recognized that reactive oxygen species (ROS) are formed during the reperfusion of ischemic tissues and ROS may be pathogenic in adult tissues. Although there is little information on the formation and toxicity of ROS during prenatal life, a strong association has been made between limb and possibly brain malformations and uteroplacental ischemia during fetal stages of gestation. It has been proposed that these malformations result from attack by ROS formed during the resumption of placental perfusion. Studies reported here examined formation of ROS in teratogenically sensitive limb and brain and insensitive heart before and during the period of teratogenic sensitivity. Also examined was the formation of ROS following hypoxia and reoxygenation in fetal culture and DNA hydroxylation in sensitive and insensitive fetal tissues during uteroplacental ischemia and reperfusion in vivo. Rates of formation of superoxide anion radical and hydrogen peroxide declined with increasing gestational age whereas those for hydroxyl radical increased. Hydrogen peroxide generation was greatest in insensitive heart whereas hydroxyl radical formation was significantly lower in brain than in limb or heart, which had comparable rates. Hydrogen peroxide generation, which declined significantly in fetuses, but not in membranes with gestation, failed to respond to reoxygenation in vitro. Finally, there were significant increases in DNA hydroxylation in fetal hearts and limbs, but not in brains during uteroplacental ischemia but no further significant change could be detected after reperfusion.

The effect of raw wheat bran, alfalfa meal and alpha-cellulose on iron ascorbate chelate and ferric chloride in three binding solutions.

Iron ascorbate chelate was prepared and purified with iron and ascorbate combined in a 1:1 molar ratio. Some iron dissociated from the chelate and bound to raw wheat bran and alfalfa meal, indicating something in these fiber sources produced the dissociation. The chelate bound intact to alpha-cellulose in bicarbonate and phosphate buffer. More iron as ferric chloride bound to the three fiber sources than did iron as chelate. Iron became more soluble in bicarbonate buffer in the presence of raw wheat bran and alfalfa meal, but became less soluble in phosphate buffer in the presence of these fiber sources.

Fetal iron balance in the rat.

Maternal and fetal iron balance through pregnancy was examined in the rat. The 20th day was selected for detailed study because of the peak iron requirements at that time. The standard diet provided a borderline iron supply to the fetus due to the limited availability of its iron for absorption. When a more available form of iron was used, normal fetal development occurred over a range of dietary iron content from 16 to 2500 mg/kg. At a level of 5 to 8 mg/kg, there was attrition of placental tissues with frequent fetal death and resorption. When the iron-deficient pregnancy was sustained, both maternal and fetal iron deficiency were present. At progressively higher levels of dietary iron, feto-placental iron content was constant despite a progressive increase in maternal iron stores. Fetal iron supply appeared to be determined primarily by plasma iron concentration, and, at normal levels, about 25% of transferrin iron passing through the uterine vasculature, was removed by the intact placentas. Low levels of plasma iron resulted in damage to fetal tissues and reduced the capacity of placental tissues to take up iron. At high levels of plasma iron, plasma iron turnover initially increased 5-fold over basal levels in nonpregnant animals due to increased placental uptake. However, with continued hyperferremia, uptake was regulated so as to maintain fetal iron at a normal level. A comparison of these data with human iron requirements explained the occurrence of both maternal and fetal iron deficiency in the rat, but only maternal iron deficiency in the human.

Maternal barbiturate administration and offspring response to shock.

Gravid Sprague-Dawley-derived rats were injected SC twice daily with either 20 or 40 mg/kg pentobarbital sodium (PT), sodium phenobarbital (PH), or the same volume of the saline vehicle on days 9-21 of pregnancy. Pair-feeding was employed. Vital, developmental, and activity measures were obtained on the neonates and locomotor activity was measured from 3-10 months of age. Avoidance was measured sequentially in a shuttle box, and in an operant chamber beginning at 3 months of age. The PH-80 dams gained less weight over the gestational period, and PH-80 and PH-40 offspring had more neonatal deaths. These male offspring were hyperactive at maturity, and PH-80 rats were initially slower to escape experimenter-initiated shock. PT exposure caused transient neonatal and juvenile hyperactivity. PT rats performed more poorly on both the conditioned avoidance and Sidman shock schedules, and had significantly lower brain: body weight ratios at 1 year of age. All four drug groups outperformed the saline offspring on subject-initiated shock schedules (punishment). Sex of offspring was determined on postnatal day 4 and the sex ratio was shifted towards male births with both drugs relative to controls.

Studies of the cellular distribution of superoxide dismutases in adult and fetal rat tissues.

Activities of three types of superoxide dismutase in tissue fractions were significantly lower in fetal and adult brain and fetal limb preparations than in fetal and adult heart preparations. An exception was the cytoplasmic fraction of adult brain that had levels of Cu, Zn-superoxide dismutase activity comparable to those in cytoplasmic fractions of heart. In addition, Mn superoxide dismutase activity appeared to be very low in all fetal mitochondrial matrix fractions and cytoplasmic fractions as well as in adult brain. Finally, the results of these studies emphasize the importance of two antioxidant defense systems in the tissues studied, one associated with the mitochondrial electron transport system and the other, the cytosolic Cu, Zn enzyme.

IgG subclasses in human periodontal disease: III. Serum concentrations of IgG subclass immunoglobulins and circulating immune complexes.

No quantitative changes in serum IgG subclass concentrations could be correlated clinically with periodontal disease, despite evidence of restrictive IgG sublcass antibody responses in inflamed gingiva. Serum from periodontal patients also lacked circulating immune complexes.

Isolation and characterization of inflammatory cells from the human periapical granuloma.

Twelve histologically-confirmed periapical granulomas were evaluated by conventional immunologic rosette assays for the presence of T-lymphocytes and complement receptor-bearing lymphocytes. A technique for dispersing the granuloma cells into suspensions was adopted to facilitate performance of the assays which were not applicable to tissue sections. Differential cell counts by an acridine orange vital dye method disclosed that the cell suspensions contained 30% macrophages, 44% lymphocytes, 15% plasma cells, and 12% neutrophils. Complement receptor-bearing cells comprised 17.9%, and T cells comprised 34.5% of the unseparated inflammatory cells. This study provides the first direct evidence of a predominance of thymic-derived lymphocytes in the lymphocyte compartment of the periapical granuloma. Analysis of the data shows that cell-mediated immunity most likely plays a role in the pathogenesis of the periapical granuloma.

Identification of collagenase in cultured blood mononuclear cells.

Mononuclear cells isolated from peripheral whole blood were fractionated by erythrocyte-rosetting into T cells and non-T cell subpopulations which were tested for collagenase-producing capacity. Mononuclear cells and purified T lymphocytes, as well as non-T (B) lymphocytes, produced collagenase during serum-free culture. Removal of the macrophages from the non-T cells did not affect collagenase production by lymphocytes.

Quantitative distribution of inflammatory cells in recurrent aphthous stomatitis.

Histologic quantitation of leukocytes in biopsies of recurrent aphthous ulcers revealed at least two morphologically-distinct inflammatory infiltrates. Lymphocytes were found consistently in pre-ulcerative lesions and in the expanding margins of the developing ulcer. In contrast, polymorphonuclear leukocytes predominated only in areas of frank ulceration.

Human gingival lymphocytes. I. Methodology for the isolation of human gingival lymphocytes.

Various methodologies were examined for the isolation of inflammatory cells from diseased human gingiva. The best recovery of viable gingival lymphocytes (gMNC) was achieved by a method which combined initial collagenase digestion followed by gentle teasing with an 18-gauge needle.

Comparison of the plaque microflora in immunodeficient and immunocompetent dental patients.

The nature and extent of the immune dysfunctions in 20 immunodeficient patients, as well as the immunocompetence of 22 control subjects, were verified by cell-mediated responsiveness and immunoglobulin quantitations. Comparisons of the microbial composition of supragingival plaque between the two populations showed that a greater number of immunodeficient than control subjects harbored Candida sp. and Staphylococcus sp. Conversely, a lower number of immunodeficient than control subjects harbored Streptococcus mutans. Also, patients with immune dysfunctions had a lower dental caries experience than their immunocompetent counterparts.

Honey bee venom melittin: correlation of nonspecific inflammatory activities with amino acid sequences.

The nonspecific (nonallergic) inflammatory activity of melittin, a protein toxin from honeybees, was correlated with specific amino acid sequences. Two different amino acid sequences of melittin were found to contribute to nonspecific inflammatory activities in guinea pig skin. Carboxy terminal peptide sequences of 7--10 amino acids induced immediate inflammatory reactions that reached maximum intensities by 15--30 min, then rapidly dissipated. The amino (N) terminal hydrophobic sequence, although not directly inflammatory, intensified the immediate reaction, causing a severe lesion evident by 2 h and characterized by massive polymorphonuclear leukocyte infiltration. A conceptual model of bee venom-induced inflammation in nonallergic individuals is suggested.

Immunologic study of human periodontosis (juvenile periodontitis).

This study was designed to define the relative distribution of lymphocytes (Lc) and plasma cells (Pc) and the nature of cell-associated immunoglobulins (Ig), IgG, IgM, IgA, IgD, and IgE, and IgG subclasses IgG1, IgG2, IgG3 and IgG4, in gingiva from patients with periodontosis (juvenile periodontitis). The patient population consisted of nine persons, eight males and one female between the ages of 18 and 20. Gingival tissues were obtained from the interproximal area associated with either the maxillary or mandibular first molar and the adjacent premolar (autologous control). Histologic and immunofluorescent quantitation of Ig bearing Lc and Ig positive Pc was assessed using 10 consecutive serial sections (3-4 micrometer) from each specimen. Periodontosis lesions were associated with pockets ranging from 6.0 to 11.0 mm and exhibited a mean G.I. = 0.5 Plasma cells were the predominant inflammatory cell present. The quantity and distribution of inflammatory cells increased with clinical severity. Autologous gingival biopsies from areas with a G.I. - 1.0, and pocket depths equal to a mean = 2.6 mm, contained numerous Lc and Pc. Seventy-six percent of the Pc in periodontosis lesions lacked demonstrable cytoplasmic heavy chain Ig determinants typical of such cells. The predominant Ig staining cells present were IgG followed by IgA and IgM. Autologous gingival biopsies contained predominantly Ig-negative Lc and very few Ig-positive Pc. Plasma cells in periodontosis lesions exhibited marked reactivity to kappa and lambda anti-light chain antiserum. The absence of heavy chains in Pc in periodontosis lesions may be another manifestation of the overall pattern of cellular and humoral abnormalities in periodontosis patients.

Periodontal status of patients with abnormalities of the immune system. II. Observations over a 2-year period.

Patients with IgA deficiency and agammaglobulinemia were pair-matched to immunocompetent subjects by age and Plaque Index. Clinical examinations performed initially and after 2 years included the Plaque Index, Gingival Index, Periodontal Disease Index, caries experience (DMF-T) and full mouth radiographic surveys. Therapy during the 2-year period included oral hygiene instructions, prophylaxis and indicated restorative treatment. Immunodeficient patients manifested consistently lower levels of gingival inflammation than matched immunocompetent patients at both examination periods. Caries experience of immunodeficient patients was also less during the study interval, and five of these patients demonstrated DMF-T scores of zero. No developmental abnormalities, acute gingival or mucosal lesions, or attachment loss associated with periodontitis was observed in either group.