The Effect of Potassium Concentration on Glycolysis in High and Low Potassium Dog Erythrocytes

The effect of potassium concentration on glycolysis was studied in the erythrocytes of Japanese Shiba dogs with high potassium (HK) and low potassium (LK) concentrations. When intracellular Na and K concentrations were changed in intact erythrocytes, lactate formation increased with increasing K concentration in both HK and LK dogs. In the reconstituted haemolysates, lactate formation, phosphofructokinase (PFK) and pyruvate kinase (PK) activities increased with increasing K concentration in both HK and LK dogs. These results suggest that glycolysis in erythrocytes of HK and LK dogs is dependent on K concentration because PFK and PK require K for their activities. In the reticulocytes of a LK dog possessing high K and low Na concentrations, PK and PFK activities were markedly elevated from the levels in mature erythrocytes and also dependent on K concentration. As in HK dog erythrocytes, high K concentration is required for glucose metabolism in LK dog reticulocytes. It is suggested that the glycolytic system of LK dog erythrocytes retains the potential to be stimulated with high K concentration even after cell maturation.     

Comparative Biochemistry of Marsupial Erythrocytes: A Review

Although extensive studies have been performed on human erythrocytes, there is a shortage of information on marsupial erythrocytes. Studies on haematology and biochemistry are useful in the ecomanagement of these animals especially those in wildlife parks and zoos. The present review summarises our findings from ∼30 species of marsupials. As marsupials show great diversity in physical and behavioural characteristics, it is not surprising that examination of their red blood cells reveals variation in the biochemical features. Many variations in red cell biochemistry appear to be species specific and, although interesting, are too numerous to list here and have been discussed in the relevant sections. Red blood cells of several species of marsupial differ from those in placental mammals by having higher haemoglobin concentration and haematocrit, lower ATP and higher 2,3-diphosphoglycerate concentrations. These features are consistent across the majority of marsupial species, but the relevance of these variations from red cell metabolism in the placental mammals is unknown.     

Methaemoglobin Formation in Euros and Bettong

Oxidant sensitivity was investigated in erythrocytes from Barrow Island euro (Macropus robustus isabellinus), mainland euro (Macropus robustus erubescens) and brushtailed-bettong (Bettongia pericillata). Oxidant sensitivity was tested by measuring methaemoglobin formation in haemolysates treated with H₂O₂ or NaNO₂. Barrow Island euro produced the highest methaemoglobin formation with H₂O₂. There was a significant negative correlation between catalase activity and H₂O₂-induced methaemoglobin formation. Our results provide further evidence supporting previous findings on eight species of marsupials.     

Oxidant Defence in the Red Blood Cells of the Greyheaded Flying Fox, Pteropus poliocephalus

The red blood cells (RBC) of bats (Order Chiroptera) are morphologically similar to those of other mammals, but the suborder Megachiroptera are unique in responding to very high demands for oxygen supply, more than 30 times that of resting conditions. Although greater efficiency of these cells to transport oxygen is advantageous to the animal, it could potentially expose the RBC to higher than average risk of oxidant damage. The aim of the present study was to investigate the capacity of RBCs of Pteropus poliocephalus, the greyheaded flying fox, to defend itself against oxidant stress. RBC of flying fox and human were challenged with 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The RBC from the two species were found to have similar reduced glutathione (GSH) reduction kinetics and succumbed to haemolysis at similar rates. However, haemoglobin oxidation was much faster in the flying fox suggesting that the RBC of flying fox may not enjoy as good protection as those of humans in the event of an oxidant stress.     

Distribution of Total,Free, Peptide-bound and Protein-bound Hydroxyproline in the Erythrocytes from Different Species

We have investigated the presence of total, free, protein-bound and peptide-bound hydroxyproline (Hyp) in the erythrocytes of different mammals namely, camel, bovine, sheep, human, rabbit and rat. Sheep erythrocytes had the highest concentration of free Hyp, followed by camel, bovine, rat, human and rabbit. The concentration of peptide-bound Hyp was the highest in camel erythrocytes followed by bovine, sheep (p<0.01), rat, human and rabbit (p<0.001). However, human erythrocytes had the highest concentration of protein-bound Hyp followed by rat, bovine, camel, rabbit and sheep (p<0.05). Camel erythrocytes had the highest concentration of total Hyp followed by bovine, sheep, rat (p<0.01), human and rabbit (p<0.001). Results showed that the ratio of free to peptide-bound Hyp was significantly higher in sheep as compared to other examined species (p<0.001). These variations in Hyp distribution may result from differences in collagen structure and/or composition in these species. Alternatively, they may be due to the fact that the circulating concentration of free Hyp is controlled by liver Hyp oxidase which regulates the concentration of free Hyp in plasma.     

Effects of ascorbic acid on high GSH and normal dog erythrocytes (I)

Dog erythrocytes with high GSH and normal GSH concentrations were compared under effects of ascorbic acid. In the presence of glucose, these two types of erythrocytes showed a similar increase in intracellular ascorbic acid without change in GSH levels. Addition of iron (Fe³⁺) to the incubation medium enhanced ascorbic acid accumulation. In the absence of glucose, GSH fell markedly in both types of erythrocyte and less ascorbic acid accumulated in normal GSH cells than high GSH cells. With iron, normal cells showed GSH depletion and marked methaemoglobin formation. Lipid peroxidation with ascorbic acid and iron increased at a similar rate in both types of erythrocyte and was inhibited by catalase. Ferricyanide reduction in both erythrocytes loaded with ascorbic acid were similar and increased with glucose or catalase. There was a high correlation between intracellular ascorbic acid content and ferricyanide reduction. These results suggest that high GSH and normal GSH dog erythrocytes have a similar capacity for accumulating ascorbic acid and for reducing ferricyanide. However, normal GSH erythrocytes are more susceptible to the oxidant effect of ascorbic acid than high GSH cells; this is probably due to a smaller GSH reserve, which is exhausted more rapidly under oxidative stress.     

A Study of the Distribution of Total, Free, Peptide-bound and Protein-bound Hydroxyproline in the Plasma of Arabian Camel ( Camelus dromedarius )

This study was conducted to determine the presence of total, free, protein bound and peptide bound hydroxyproline (Hyp) in the plasma of the Arabian camel (Camelus dromedarius) as compared to the sheep. Results showed that the Hyp pool of sheep plasma was composed of free, protein bound and peptide bound hydroxyproline. On the other hand, only free and protein bound Hyp contributed to the Hyp pool of the camel plasma; peptide bound Hyp was not detected. Camel plasma showed a significantly lower content of total Hyp (p<0.001), however, a higher level (p<0.001) of protein bound Hyp as compared to sheep. The observed variations in Hyp distribution may result from differences in the Hyp derivatives and/or compositions in these species. This is the first report describing the distribution of Hyp in camel plasma.     

Glutathione (GSH) reduces the open probability of mechanosensitive channels in Escherichia coli protoplasts

The effects of glutathione (reduced GSH, and oxidized GSSG) on mechanosensitive (MS) ion channels from Escherichia coli protoplasts were investigated using excised, inside-out membrane patches. Our studies demonstrate here that 5 mM GSH irreversibly reduces the activities of the 560-pS MS channel by approximately 70–75% whereas 5 mM GSSG did not alter the MS channel currents. In addition, millimolar concentrations of dithiothreitol had similar although weaker effects to GSH. The physiological concentration of GSH in E. coli cytoplasm ranges from 3.5 mM to 6.6 mM, which may indicate that under normal conditions these MS channels open less due to membrane stretch. Received: 26 January 1999 / Received after revision: 18 March 1999 / Accepted: 19 March 1999     

Relationship Between the Reference Value of the Erythrocyte Sedimentation Rate of the Presenile Population and of Geographical Factors in China

In order to provide a scientific basis for a strictly medical comparison (a ‘unified standard’) of local reference values for erythrocyte sedimentation rate (ESR) among the presenile population in China, the reference values for ESR of healthy persons have been collected, the assays having been performed by the method of Wintrobe. The relationship between the reference value for ESR in the presenile population and geographical factors has been examined. Altitude was found to be the most important factor affecting the reference value for ESR in the presenile population; ESR reference values decrease as the altitude gradually increased, the relationship being highly significant. The method of stepwise regression analysis was used to deduce two regression equations, so that if the geographical factor value of a particular area in China is known, the national reference value for ESR in the presenile population can be calculated by means of these equations. Based on geographical factors, China can be divided into six districts: Qingzang, Southwest, Northwest, Southeast, North and Northeast.     

A Comparison of the Effects of Repeated Anaesthesia with Ether or Isoflurane in Rats

Ether anaesthesia has been used in rodents for routine blood sampling in toxicological studies for many years. Because of the highly flammable nature of ether and the inability to control the depth of anaesthesia, more suitable alternatives are needed. This study was conducted to compare data obtained using ether or isoflurane. Groups of male and female Sprague–Dawley rats, 5 weeks of age, were maintained on a normal diet for up to 25 weeks. Body weights were recorded during the study. Blood was sampled from the retro-orbital sinus under the different anaesthetics from the animals at 8, 12, 21 and from females only at 25 weeks of age for the analysis of standard haematology and biochemical parameters. These animals were killed at 25 weeks of age for histological examination. Body weights were unaffected. The major biochemical and haematological parameters were similar at the various time points with both types of anaesthetic. Minor variations, such as an increase in potassium and a reduction in glucose with isoflurane were seen, but there was no consistent trend. Examination of the liver, kidneys, heart and lungs did not reveal any changes that were considered to be associated with either anaesthetic. Blood was taken for prolactin analysis from a subgroup of animals at 12 weeks of age anaesthetised with ether or isoflurane, and from tail-bled animals without anaesthesia, these animals being killed at 17 weeks of age for histological examination. The level of prolactin at 12 weeks of age was reduced in the isoflurane group particularly in the males. This is considered to indicate that there was less stress. Based on these results, isoflurane is proposed as a suitable alternative to ether.     

Comparison of Glutathione S-Transferase and Alanyl Aminopeptidase as Viability Markers in a Porcine NHBD Model

A critical shortage in the kidney donor pool has led to a renewed interest in non-heart beating donors (NHBDs). As warm ischaemic tissue injury is a characteristic of NHBD, it is therefore crucial to screen such kidneys for viability to disqualify kidneys that will never work, i.e. develop primary non-function. Viability screening is somewhat time-consuming, usually involving a combination of donor history, visual assessment of the donor kidney at procurement and monitoring during hypothermic machine preservation. Machine perfusion of NHBD kidneys enables physical characteristics to be evaluated by perfusion flow rate and pressure. Glutathione S-transferase (GST) is an established kidney injury biomarker, but its prolonged assay time may result in delays in any potential transplantation. Alanyl aminopeptidase (Ala-AP) has been developed as an alternative novel biomarker of ischaemic renal injury. A porcine NHBD model with differential ischaemic renal injury, using heparin ± streptokinase in the initial in situ flush solution, was developed to evaluate the two biomarker enzymes, GST and Ala-AP. Two groups of 16 porcine NHBD kidneys (heparin alone or heparin + streptokinase) were machine perfused for 4 h, with documentation of pressure, flow, resistance and temperature and samples of kidney perfusate were taken at hourly intrevals. Wedge sections of the kidneys were analysed at termination of perfusion. Histopathology of renal wedge sections, machine perfusion physical parameters and biomarkers demonstrated the differential renal ischaemic insult. The two biomarkers, GST and Ala-AP appear to be correlate with the degree of ischaemic injury and with each other. Mild ischaemic injury occurs in the streptokinase-treated group, with lower efflux of GST and Ala-AP. Assays for Ala-AP activity in humans are significantly shorter than the comparative assays for GST, enabling early decision making on viability. This can, therefore, facilitate transplantation of the potential NHBD kidney within the recommended 24-hour period of cardiac arrest of donor.     

Effects of Duration of Storage and Storage Temperature on Cell Counts of Bovine Blood Samples as Determined by an Automated Haematology Analyser

Analysis of blood cells is an important part of many scientific investigations in the field of cattle herd health. Over the last 30 years, automated blood analysis has all but replaced manual counting of blood cells using counting chambers. The present study investigated the effects of prolonged storage and storage temperatures on cell counts as determined by a haematology analyser. Blood samples from 20 clinically healthy cows were repeatedly analysed with a Cell-Dyn 3500 (Abbott Diagnostika, Delkenheim), within 24 hours after collection and after storage at either 4° C or 20° C. The counts of most blood cells were more stable in samples stored at 20° C than those stored at 4° C. For at least 8 h, the counts of all analysed cell types, with the exception of lymphocytes, remained within ±3 standard deviations that were calculated for fresh samples, provided that the blood was stored at 20° C. Correspondence and offprint requests to: Dr Ulrich Bleul, Klinik für Fortpflanzungskunde, Universit?t Zürich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.     

Studies on the Relationship Between Haemoglobin Types of Adult Dromedary Camels and the Concentrations of Haemoglobin, Copper, Ceruloplasmin and Iron

The relationship between haemoglobin types of adult dromedary camels and the concentrations of haemoglobin, copper, ceruloplasmin and iron were studied. Blood samples were taken from the jugular vein of 100 clinically healthy Iranian dromedary camels according to their age (1–2, 2–4, 4–6 and >6 years) and sex. Haemoglobin electrophoresis showed two haemoglobin types: HbA (95.2%) and HbA₂ (4.8%). Age and sex had no significant effect on haemoglobin types nor on the concentrations of haemoglobin, copper, ceruloplasmin and iron. There was no correlation between the concentrations of haemoglobin, copper, iron, ceruloplasmin, HbA and HbA₂.     

Effect of a Selenium-Deficient Diet on Blood Values of T3 and T4 in Cows

Pastures in the south of Chile have been shown to be selenium deficient, and as selenium is a component of deiodinase type 1, which is necessary for the synthesis of triiodothyronine (T3) at peripheral levels, the effect of selenium deficiency on the blood concentration of theses hormones in cows was studied. Twelve pregnant Friesian cows were randomly allocated into two homogeneous groups of six animals each - selenium supplemented and selenium deficient. The selenium deficient diet consisted of 11.5 kg of hay, 500 g of soya bran, 150 g of mineral mix (without selenium), 500 g of fat for animal feeding, and was given from day 15 of lactation until the end of the study. A commercial concentrate (Cosetan) and urea was also given according to lactation requirements (up to 5 kg and up to 120 g, respectively). The selenium content was 0.05 ppm of dry matter (equivalent to 18% of the daily requirements). The supplemented group was injected subcutaneously with 1 mg Se/kg, using the commercial product Deposel (1 ml/50kg). Blood samples were obtained by coccygeous vein venepuncture before supplementation (prepartum basal values) and thereafter every 15 days. GSH-Px activity in plasma was measured using a HITACHI 4020 spectrophotometer. Serum T3 and thyroxine (T4) concentrations were measured by electrochemiluminiscence. ANOVA, Tuckey test and Student’s t-test were used to establish the significance of the intra group differences, with p=<0.05 being considered significant. There were no significant differences in blood values of T4 in either the supplemented or non-supplemented groups during pregnancy and lactation. However the blood values of T4 decreased significantly (p=<0.05) during days 30–60 of lactation to values below the reference range. The T3 serum concentrations in the supplemented group of cows were significantly lower (p=<0.05) at 60, 90 and 150 days of lactation than cows that had been supplemented with selenium.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant (Elephas maximus) and Man (Homo sapiens)

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

Mathematical Model of Time Needed for the Immune System to Detect and Kill Cancer Cells in Blood

Disfunction of the immune system has generally been blamed for the development of cancers however, the immune system needs time to detect and kill cancer cells. To date, the chance of random collision between immune cells and cancer cells in blood has drawn less attention. In this study we used a random principle to analyse the possibility of collision between immune and cancer cells in blood. With the criterion of p>0.95, the results show that an immune cell needs, for example, five random collisions in order to hit a cancer cell when there are one cancer cell, one immune cell and one normal cell in our consideration; and 14 999 999 random collisions in order to hit a cancer cell when there are one cancer cell, one immune cell and 4 999 999 normal cells in consideration. Furthermore, we analyse how different proliferation rates of cancer cells affect the random collisions between the detecting/killing immune cells and cancer cells.     

The effects of changes to action potential duration on the calcium content of the sarcoplasmic reticulum in isolated guinea-pig ventricular myocytes

. We have estimated sarcoplasmic reticulum calcium content using rapid application of caffeine on voltage clamped, isolated guinea-pig ventricular myocytes. Caffeine induces the release of calcium from the sarcoplasmic reticulum and this calcium is extruded from the cells by the sarcolemmal Na/Ca exchange. Integrating the inward Na/Ca exchange current thus allows estimations of sarcoplasmic reticulum calcium content. Ventricular myocytes were stimulated to reach new steady-states by action potential voltage clamps of varying duration. Once contractile steady-state had been reached caffeine was rapidly applied in place of the next action potential and sarcoplasmic reticulum calcium content measured. Prolonging the action potential duration increased sarcoplasmic reticulum calcium content and vice-versa. This calcium loading may underlie the positive inotropic effect of increased action potential duration. Received: 11 July 1996 / Received after revision: 15 October 1996 / Accepted: 26 November 1996     

Comparative Nuclear Magnetic Resonance Studies of Diffusional Water Permeability of Red Blood Cells from Different Species. XI. Horses Introduced to Australia and European Horses ( Equus caballus )

The diffusional water permeability (P _d) of red blood cells (RBCs) from horses introduced to Australia and from European horses was measured by a Mn²⁺ doping nuclear magnetic resonance (NMR) technique. The values of P _d were ∼3.5 × 10⁻³ cm/s at 25°C, 4.1 × 10⁻³ cm/s at 30°C, 5.6 × 10⁻³ cm/s at 37°C and 6.3 × 10⁻³ cm/s at 42°C with no significant differences between the two strains of horse. Systematic studies on the effect of p-chloromercuribenzene sulphonate (PCMBS) on water diffusion indicated that the maximal inhibition (60%) was reached in 60 min at 22°C with 1 mm PCMBS. The basal permeability to water was estimated at 1.2 × 10⁻³ cm/s at 25°C, 1.7 × 10⁻³ cm/s at 30°C, 2.0 × 10⁻³ cm/s at 37°C and 2.7 × 10⁻³ cm/s at 42°C. The activation energy (E _a,d) of water diffusion was ∼25 kJ/mol and increased to ∼39 kJ/mol after incubation with PCMBS, in conditions of maximal inhibition of water diffusion. The membrane protein electrophoretic pattern of horse RBCs was compared with its human counterpart. The horse RBC membranes had lower amounts of the proteins migrating as bands 4.1 and 4.2 and higher amounts of the protein migrating as band 4.9, and band 6 (glyceraldehyde-3-phosphate dehydrogenase) was undetectable. A noteworthy feature was the appearance of considerable differences in protein migration distances in the region of bands 7 and 9, between horse and human membranes.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant ( Elephas maximus ) and Man ( Homo sapiens )

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

Effects of magnetic fields on the accumulation of thiobarbituric acid reactive substances induced by iron salt and H2O2 in mouse brain homogenates or phosphotidylcholine

In this study, we examined the effects of magnetic fields (MFs) on the generation of thiobarbituric acid reactive substances (TBARS) in the mouse brain homogenates or phosphotidylcholine (PC) solution, incubated with FeCl₃ and/or H₂O₂. Active oxygen species were generated and lipid peroxidation was induced in mouse brain homogenates by incubation with iron ions, resulting in the accumulation of TBARS. Lipid peroxidation was induced in PC by incubation with iron ions and H₂O₂. Exposure to sinusoidal MFs (60 Hz, 0.2–1.2 mT), symmetric sawtooth-waveform MFs (50 Hz, 25–600 mT/s), rectangular MFs (1/0.4–1/16 Hz, 3.3 mT) and static MFs (1, 5–300 mT) had no effect on the accumulation of TBARS in brain homogenates induced by FeCl₃. In contrast, when the homogenates were incubated with FeCl₃ in static MFs (2–4 mT), the accumulation of TBARS was decreased. However, this inhibitory effect disappeared when EDTA was added to the homogenate and incubated with H₂O₂. The accumulation of TBARS in PC solution incubated with FeCl₃ and H₂O₂ was also inhibited by the static MF. These results indicate that only static MFs had an inhibitory effect on iron-induced lipid peroxidation and the effectiveness of this magnetic field on iron ion-induced active oxygen species generation is restricted to a so called ‘window’ of field intensity of 2–4 mT.