John W. Harvey

The Erythrocyte: Physiology, Metabolism, and Biochemical Disorders

Publisher Summary This chapter discusses the physiology, metabolism, and biochemical disorders of erythrocytes. Mammalian erythrocytes or red blood cells (RBCs) are anucleate cells that normally circulate for several months in blood despite limited synthetic capacities, and repeated exposures to mechanical and metabolic insults. The primary purpose of RBCs is to carry hemoglobin (Hb), a heme-containing protein that accounts for more than 90% of the protein within RBCs. Most RBCs in normal dogs, cats, horses, cattle, and sheep occur in the shape of biconcave disks (discocytes). The degree of biconcavity is most pronounced in dogs, and less so, in cats and horses. RBCs from goats generally have a flat surface with little surface depression; a variety of irregularly shaped RBCs (poikilocytes) may be present in clinically normal goats. This chapter begins with a discussion on differences in erythrocyte shape and functions of RBCs. The chapter then elaborates concepts related to hematopoietic microenvironment and hematopoietic growth factors. Biochemistry of iron metabolism, Hb synthesis, and reticulocytes are also discussed. The chapter explains in detail the membrane structure of RBCs, as well as concepts related to shape and deformability, membrane transport, and carbohydrate metabolism. The chapter concludes with discussing inherited disorders of RBCs.

Studies of the efficacy and potential hazards of methylene blue therapy in aniline-induced methaemoglobinaemia

Summary. The similarity between poison and antidote was known to the ancient Greeks who used the same word, pharmakon, for both. This paper presents evidence that aniline (the toxin) and methylene blue (the therapy) are in fact remarkably similar and additive in some of their effects on erythrocytes. Studies were prompted by a case of aniline‐induced methaemoglobinaemia in which two injections of methylene blue did not rapidly eliminate cyanosis and were followed by severe, delayed haemolysis. Interactions between aniline and methylene blue were studied in cats which, although showing important differences from man in their haemoglobin and splenic vasculature, represent a useful model. Methylene blue potentiated the oxidative denaturation of haemoglobin by aniline as judged by the size and number of Heinz bodies and their turbidity in haemolysate. It also aggravated and prolonged the fall in erythrocyte reduced glutathione content which occurred at a time of maximum Heinz body production. While methylene blue in judicious dosage will reduce the content of methaemoglobin after aniline exposure, it may not eliminate visible cyanosis. Repeated injections of methylene blue can markedly aggravate subsequent haemolysis without further lowering methaemoglobin content.

COMPARISON OF METHODS USED TO DIAGNOSE GENERALIZED INFLAMMATORY DISEASE IN MANATEES (TRICHECHUS MANATUS LATIROSTRIS)

Abstract Manatees (Trichechus manatus latirostris) are afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as “cold stress syndrome” and pneumonia. White-blood-cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase-response proteins, such as haptoglobin and serum amyloid A, have proven to be sensitive measures of inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including total white-blood-cell count, albumin: globulin ratio, gel electrophoresis analysis, C-reactive protein, alpha1 acid glycoprotein, haptoglobin, fibrinogen, and serum amyloid A. Samples were collected from 71 apparently healthy and 27 diseased animals during diagnostic medical examination. Serum amyloid A, measured by ELISA, followed by albumin:globulin ratio, measured by plasma gel electrophoresis, were most sensitive in diagnosing inflammatory disease, with diagnostic sensitivity and specificity of approximately 90%. The reference interval for serum amyloid A is <10–50 μg/ml with an equivocal interval of 51–70 μg/ml. The reference interval for albumin:globulin ratio by plasma gel electrophoresis is 0.7–1.1. Albumin: globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology. Albumin:globulin ratio, measured by serum gel electrophoresis, has a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4–2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. The haptoglobin assay is significantly affected by hemolysis. Fibrinogen, measured by heat precipitation, has a reference interval of 100–400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.

CLINICAL BIOCHEMISTRY IN HEALTHY MANATEES (TRICHECHUS MANATUS LATIROSTRIS)

Abstract Florida manatees (Trichechus manatus latirostris) are endangered aquatic mammals living in coastal and riverine waterways of Florida and adjacent states. Serum or plasma biochemical analyses are important tools in evaluating the health of free-ranging and captive manatees. The purpose of this study was to measure diagnostically important analytes in the plasma of healthy manatees and to determine whether there was significant variation with respect to location (free-ranging versus captive), age class (small calves, large calves, subadults, adults), and gender. No significant differences in plasma sodium, potassium, bilirubin, glucose, alanine aminotransferase, or creatine kinase were found among these classes of animals. Compared to free-ranging manatees, captive animals had significantly lower mean concentrations of plasma chloride, phosphate, magnesium, triglycerides, anion gap, and lactate. Captive manatees had significantly higher mean values of total CO2, calcium, urea, creatinine, alkaline phosphatase, γ-glutamyltransferase, total protein, albumin, and albumin/globulin ratio than did free-ranging animals. Differences in the environments of these two groups, including diet, temperature, salinity, and stress, might account for some of these results. The higher plasma lactate and anion gap concentrations and lower total CO2 concentrations of free-ranging manatees were probably due to greater exertion during capture, but the lack of elevated plasma creatine kinase activity relative to captive animals indicates that there was no serious muscle injury associated with capture. Plasma phosphate decreased and total globulins increased with age. Plasma cholesterol and triglyceride concentrations were highest in small calves. Plasma aspartate aminotransferase was higher in large calves than in adults and subadults, and the albumin/ globulin ratio was higher in subadults than in adults. Plasma total CO2 was higher and chloride was slightly lower in females than in males.

Mammalian erythrocyte metabolism and oxidant drugs

Abstract The addition of methylene blue to erythrocyte suspensions from horse, cat, dog, and man resulted in increased glucose utilization concomitant with a marked increase in pentose phosphate pathway metabolism in each species. With the addition of ascorbate, an increase in erythrocyte glucose utilization was observed only in the horse. Erythrocyte pentose phosphate pathway metabolism was higher in man than in animals in the presence of ascorbate, suggesting that animal erythrocytes might be less able to protect themselves against peroxides and/or superoxide. Differences in superoxide dismutase activity did not appear to account for this lower metabolism in animal erythrocytes, since similar activities were measured in hemolysates prepared from each species. When blood was incubated with ascorbate and cyanide in a visual screening test designed for glucose-6-phosphate dehydrogenase deficiency in man, hemoglobin in horse and dog blood was oxidized more rapidly than human hemoglobin, whereas hemoglobin in cat blood was oxidized less readily than that in human blood. It is suggested that the lower ascorbate-stimulated pentose phosphate pathway metabolism in cat erythrocytes relative to man reflects a decreased incorporation of ascorbate intracellularly rather than a decreased ability of cat erythrocytes to respond to peroxides.

Quantitative determinations of normal horse, cat, and dog haptoglobins

Abstract A haptoglobin assay method, based on the principle that cyanmet-hemoglobin is protected from acid denaturation when bound to haptoglobin, was evaluated and found to be well suited for haptoglobin determinations in horses, cats and dogs. Wide haptoglobin ranges were observed in each species. Inasmuch as no significant differences were observed between species, a normal range of 20 to 190 mg cyanmethemoglobin binding capacity/dl serum or plasma is recommended for use in all 3 species.

Hematology of healthy Florida manatees (Trichechus manatus)

BACKGROUND Hematologic analysis is an important tool in evaluating the general health status of free-ranging manatees and in the diagnosis and monitoring of rehabilitating animals. OBJECTIVES The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender. METHODS Blood was collected from 55 free-ranging and 63 captive healthy manatees. Most analytes were measured using a CELL-DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations. RESULTS Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free-ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals. CONCLUSIONS Hematologic findings were generally similar between captive and free-ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals.

Iron Metabolism and Its Disorders

Publisher Summary This chapter explains the basic concepts related to metabolism of iron and disorders associated with it. Iron is an essential nutrient required in a wide variety of metabolic processes. In solution, iron exists in two oxidation states. Iron is absorbed from the diet in the small intestine and transferred to plasma, where it is bound to transferrin for transport to cells within the body. Once inside the body, iron cycles in a nearly closed system because little iron is lost in domestic animals unless hemorrhage occurs. About 75% of the iron present in plasma is transported to the bone marrow for incorporation into hemoglobin in developing erythroid cells. The remaining plasma iron is taken up by nonerythroid tissues, primarily the liver. Erythrocytes containing hemoglobin normally circulate for several months before being phagocytized by macrophages when senescent. After phagocytosis, erythrocytes are lysed, hemoglobin is degraded, and iron is released.This chapter explains in detail iron distribution, iron absorption, and plasma iron transport. The chapter also discusses regulation of iron metabolism, iron metabolism in cells, and tests for evaluating iron metabolism.

Chapter 7 – The Erythrocyte: Physiology, Metabolism, and Biochemical Disorders

Publisher Summary Mammalian erythrocytes or red blood cells (RBCs) are anucleate cells that normally circulate for several months in blood despite limited synthetic capacities and repeated exposures to mechanical and metabolic insults. Their primary purpose is to carry hemoglobin (Hb), a heme-containing protein that accounts for 95% of the total protein in RBCs. The benefits of having Hb contained within cells, as opposed to free in plasma, include the much slower turnover in blood, the metabolic capability of RBCs to maintain iron in Hb in the functional ferrous state, and the ability to control Hb oxygen affinity by altering the concentrations of organic phosphates (especially 2,3DPG). In addition, the presence of free Hb in plasma in concentrations normally found in whole blood would exert an osmotic pressure several times greater than that normally exerted by plasma proteins, profoundly affecting the movement of fluid between the vascular system and tissues.

Methaemoglobin reductase deficiency in dogs

Erythrocyte methaemoglobin reductase deficiency is described in a toy Alaskan Eskimo dog, a miniature poodle dog and a cocker/poodle cross dog. Blood methaemoglobin contents ranged from 19% to 36% of total haemoglobin, with methaemoglobin reductase values between 13% and 33% of normal. There appeared to be a negative linear correlation between erythrocyte methaemoglobin content and methaemoglobin reductase activity. A single intravenous injection of methylene blue (1 mg/kg body weight) resulted in a dramatic decrease in methaemoglobin content within 1 hour when given to two of the deficient dogs in the present study. Following the disappearance of methylene blue from blood, methaemoglobin content increased linearly at 3.2% and 2.5% per day in these dogs, suggesting that about 3.0% of erythrocyte haemoglobin is normally oxidised to methaemoglobin each day in dogs, as has been estimated in humans. Oral riboflavin therapy was not effective in reducing blood methaemoglobin content in deficient dogs.