Factors influencing choice between tracheostomy and prolonged translaryngeal intubation in acute respiratory failure: a prospective study

Hematologic parameters were studied in giant Canada geese (Branta canadensis maxima), mallard ducks (Anas platyrhynchos platyrhynchos) and various species of diving ducks at seasinal intervals throughout the year. Highest values for packed cell volume, hemoglobin content and erythrocyte counts were found in the winter and pre-nesting periods. Mean corpuscular volume and mean corpuscular hemoglobin varied inversely with these values.     

Characterisation of erythrocyte shapes and sizes by NMR diffusion-diffraction of water: correlations with electron micrographs

Irradiation of the mammalian foetus produces a broad spectrum of congenital abnormalities, growth retardations, developmental delays, and functional deficits, depending upon the dose and the specific gestational phase of irradiation. The developing brain is particularly susceptible to production of deleterious effects, with decreased brain size, behavioural alterations, and mental retardation having been documented. Supplementing the limited human data, rodent models have been extensively used to investigate the specific processes by which relatively low doses, with correspondingly minor cellular damage to the developing neocortex, can produce dramatic postnatal consequences in brain structure and function. The effects of a variety of physical (dose, linear energy transfer, dose rate, fractionation) and biological (species, strain, gestational age, time course post-irradiation) parameters have been examined in an attempt to provide much needed information on such critical aspects as dose response, threshold doses for effect, and extrapolation to human risk estimates. Various acute cellular responses (e.g. appearance of pyknotic cells and macrophages) observed in the developing neocortex 0-24 h after in utero irradiation can be associated with postnatal effects. Moreover, it is possible to correlate thinning of specific layers of the cerebral cortex with specific behavioural aberrations, allowing prediction of brain structural changes from functional alterations, and vice versa. Thus, it is possible to speculate as to the mechanisms and targets for extremely sensitive, radiation-induced cellular damage in the developing foetal brain, that will interfere with the orderly and precisely programmed development of the mammalian brain, leading finally to postnatal expression as delays in growth and development, perturbations in behaviour, and alterations in brain structure.     

HPLC detection of fetal blood in meconium: improved sensitivity compared with qualitative methods

We describe an HPLC-based method for the detection and quantification of fetal hemoglobin in stools of newborns. The new procedure is an alternative to the classic qualitative test for adult hemoglobin in meconium based on the differential stability of hemoglobin species in dilute base (Apt test). The HPLC method, based on a commercial device for hemoglobin characterization (Bio-Rad Variant), readily separates fetal and adult hemoglobin from non-hemoglobin components of meconium. To validate the method, blood and meconium were mixed in various proportions and then prepared for analysis with extraction in saline. The HPLC method accurately identified hemoglobin species even when the blood constituted only 5 mL per 100 g of the meconium specimen, and nearly quantitative recovery of hemoglobin was obtained at a blood content of 20 mL per 100 g of the meconium. Analysis time was 6.5 min, and preparation of sample was simple. HPLC detection of fetal blood in stools or other specimens markedly improves detection/characterization of blood in meconium.     

Erythrocyte dehydration in pathophysiology and treatment of sickle cell disease

A prominent feature of sickle cell disease is the presence of cells with markedly increased sickle cell hemoglobin concentration, as a consequence of the loss of potassium, chloride, and water from the erythrocyte. Because of the extreme dependency of the kinetic of polymerization on sickle cell hemoglobin concentration, these dehydrated erythrocytes have an increased tendency to polymerize and sickle. Thus blockade of the loss of potassium from the erythrocyte should prevent the increase in sickle cell hemoglobin concentration and reduce sickling. The availability of this potential therapeutic option is based on a detailed knowledge of the mechanisms leading to cell dehydration. Two ion transport pathways, the K-Cl cotransport and the Ca(2+)-activated K+ channel, play a prominent role in the dehydration of sickle erythrocytes. Possible therapeutic strategies include inhibition of K-Cl cotransport by increasing erythrocyte Mg2+ content and inhibition of the Ca(2+)-activated K channel by oral administration of clotrimazole.     

Nonuniform biosynthesis of multiple hemoglobins in the adult rat and guinea pig

Separation of adult rat bone marrow cells by the method of thin layer countercurrent distribution permits the analyses of 59Fe-tagged erythroid cells for the various multiple hemoglobins and the assignment of such hemoglobins to erythroid cells at different stages of their development. Of the six adult red cell hemoglobins, hemoglobin 5 is synthesized most actively in the earliest erythroid cell whereas hemoglobin 4 (the major hemoglobin of the red cell) is synthesized most actively in the latest erythroid cells, e.g. the reticulocyte. Experimental evidence also indicates that maturation of the erythroid cell is accompanied by a decreased rate of synthesis of hemoglobin 5. The earliest erythroid cells of the marrow contain two hemoglobins, 7 and 8, which are absent in the adult red cell. Similar studies with the guinea pig confirm the nonuniform biosynthesis of its two hemoglobins and suggest that the phenomenon may be a general one among mammalian multiple hemoglobins.     

Isolation of a non-sedimenting membrane fraction from the water soluble fraction of bovine lens

A membrane fraction was isolated from the water soluble fraction of bovine lens by increasing the density of the water soluble fraction with KBr and subjecting it to overnight centrifugation at 100000 g. We have called this fraction, which floats to the top of the mixture upon centrifugation, the non-sedimenting membrane fraction (NSMF). Electron microscopy of the NSMF revealed that it is composed of the expected membrane structures of unit membrane, fiber junction and cytoskeleton. Significantly less of the total membrane of the NSMF was devoted to fiber junction (22.8%) than in the sedimenting membrane fraction (SMF) (41.1%) prepared by sucrose density centrifugation of the water insoluble fraction. The NSMF accounted for about 7-12% of the total bovine lens membrane, and preliminary experiments demonstrated that a similar fraction could be isolated from the water soluble fraction of lenses from rats, rabbits, chickens and humans. The NSMF contained about 0.9 mg total lipid per mg total membrane protein, which was significantly greater than the value obtained for the SMF (0.5 mg total lipid per mg total membrane protein). The greater relative amount of total lipid in the NSMF was due to a significantly greater relative amount of phospholipid in the NSMF which was further reflected by the observation that the cholesterol: phospholipid molar ration of the NSMF (0.58) was significantly less than that of the SMF (0.88). Thus the relative lipid composition of the NSMF was significantly different than that of the SMF. Although the phospholipid content of the NSMF was greater than that of the SMF, the compositions of the phospholipids in the two membrane fractions were similar. The NSMF possessed essentially the same polypeptides (both extrinsic and intrinsic) which were found in the SMF. The NSMF was found to be distributed throughout the lens in a proportionate manner. We conclude that the NSMF may account for most of the lipid which remains in the water soluble fraction of the normal bovine lens after sedimentation of the water insoluble fraction. This membrane fraction substantially differs from the SMF in terms of structure and relative lipid composition. We speculate that the NSMF may represent a specialised domain of the fiber cell plasma membrane which has been previously unrecognized.     

A mechanistic view of the non-ideal osmotic and motional behavior of intracellular water

It is commonly assumed that essentially all of the water in cells has the same ideal motional and colligative properties as does water in bulk liquid state. This assumption is used in studies of volume regulation, transmembrane movement of solutes and electrical potentials, solute and solution motion, solute solubility and other phenomena. To get at the extent and the source of non-ideally behaved water (an operational term dependent on the measurement method), we studied the motional and colligative properties of water in cells, in solutions of amino acids and glycine peptides whose surface characteristics are known, and in solution of bovine serum albumin, hemoglobin and some synthetic polypeptides. Solutions of individual amino acids with progressively larger hydrophobic side chains showed one perturbed water molecule (structured-slowed in motion) per nine square angstroms of hydrophobic surface area. Water molecules adjacent to hydrophobic surfaces form pentagonal structural arrays, as shown by X-ray diffraction studies, that are reported to be disrupted by heat, electric field, hydrostatic pressure and phosphorylation state. Hydrophilic amino acids demonstrated water destructuring (increased motion) that was attributed to dielectric realignment of dipolar water molecules in the electric field between charge groups. In solutions of proteins, several methods indicate the equivalent of 2-8 layers of structured water molecules extending beyond the protein surface, and we have recently demonstrated that induced protein conformational change modifies the extent of non-ideally behaved water. Water self-diffusion rate as measured in three different cell types was about half that of bulk water, indicating that most of the water in these cells was slower in motion than bulk water. In different cell types the extent of osmotically perturbed water ranged from less that half to almost all of the intracellular water. The assumption that essentially all intracellular water has ideal osmotic and motional behavior is not supported by the experimental findings. The non-ideally of cell water is an operational term. Therefore, the amount of non-ideally behaving water is dependent on the characteristics of water targeted, i.e. the measurement method, and a large fraction of it is explainable in mechanistic terms at a molecular level based on solute-solvent interactions.     

Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis

The effect of amino acid peroxides, relatively stable products of irradiation of amino acid solutions, on erythrocyte components was studied. Interaction of proline, lysine, valine, and leucine peroxides (100-300 mu M) with erythrocyte membranes brought about a decrease of membrane protein -SH group content and of activities of (Na+, K+)-ATPase and Ca2+ -ATPase, and induced aggregation of membrane proteins, due mainly to the formation of interpeptide disulfides. Interaction of amino acid peroxides with hemoglobin brought about hemoglobin oxidation to methemoglobin. The effects of amino acid peroxides are similar to those of t-butyl hydroperoxide. These results indicate that peroxides of amino acid and proteins, which can also be formed under physiological conditions, may be mediators of the cellular action of reactive oxygen species.     

Measurement of water transport during freezing in cell suspensions using a differential scanning calorimeter

A new technique using a differential scanning calorimeter (DSC) was developed to obtain dynamic and quantitative water transport data in cell suspensions during freezing. The model system investigated was a nonattached spherical lymphocyte (Epstein-Barr virus transformed, EBVT) human cell line. Data from the technique show that the initial heat release of a prenucleated sample containing osmotically active cells in media is greater than the final heat release of an identical sample of osmotically inactive or lysed cells in media. The total integrated magnitude of this difference, Deltaqdsc, was found to be proportional to the cytocrit and hence also to the supercooled water volume in the sample. Further, the normalized fractional integrated heat release difference as a function of temperature, Deltaq(T)dsc/Deltaqdsc, was shown to correlate with the amount of supercooled cellular water which had exosmosed from the cell as a function of subzero temperature at constant cooling rates of 5, 10, and 20 degrees C/min. Several important limitations of the technique are (1) that it requires a priori knowledge of geometric parameters such as the surface area, initial volume, and osmotically inactive cell volume and (2) that the technique alone cannot determine whether the heat released from supercooled cellular water is due to dehydration or intracellular ice formation. Cryomicroscopy was used to address these limitations. The initial cell volume and surface area were obtained directly whereas a Boyle-van't Hoff (BVH) plot was constructed to obtain the osmotically inactive cell volume Vb. Curve fitting the BVH data assuming linear osmometric behavior yielded Vb = 0.258V0; however, nonlinearity in the data suggests that the EBVT lymphocyte cells are not "ideal osmometers" at low subzero temperatures and created some uncertainty in the actual value of Vb. Cryomicroscopy further confirmed that dehydration was the predominant biophysical response of the cells over the range of cooling rates investigated. One notable exception occurred at a rate of 20 degrees C/min where evidence for intracellular ice formation due to a DSC measured heat release between -30 and -34 degrees C correlated with a higher end volume but no darkening of the cells during cryomicroscopy. For the cooling rate tested (5 degrees C/min) the cryomicroscopy data correlated statistically very well with the DSC water transport data. A model of water transport was fit to the DSC water transport data and the average (5, 10, and 20 degrees C/min) biophysical parameters for the EBVT lymphocytes were found to be Lpg = 0.10 micro m/min-atm, ELp = 15.5 kcal/mol. Finally, the decrease in heat release from osmotically active cells measured by the DSC during repetitive freezing and thawing was found to correlate strongly with the viability of the cells measured during identical freeze/thaw protocols with cryomicroscopy. This shows the additional ability of the technique to assess freeze/thaw injury. In summary, this DSC technique is a promising new approach for measuring water transport in cellular systems during freezing.     

Sebum and water content in the skin of aged immobilized patients

In the present study we investigated the sebum content and hydration of the skin in aged immobilized patients. Healthy aged as well as young and aged immobilized patients were evaluated by photometry, using a sebum tester and capacitance meter to detect sebum and hydration, respectively, in various skin areas. Sebum content was significantly higher in the young groups as compared to the aged ones, including those of the immobilized patients. Similar values of water content were observed in the healthy young and aged volunteers. However, surprisingly, a significantly marked decrease was detected in each tested area of the immobilized patients. We may assume that a cascade of events caused by the immobilization status of the patients leads to a significant decrease in water content within the dermis.     

Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue

We developed a new, highly sensitive enzymatic method for quantifying creatine in erythrocytes, which comprises creatine amidinohydrolase, sarcosine oxidase, and peroxidase. In the present method, an N-methylcarbamoyl derivative of methylene blue, 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP), was used as a sensitive chromogenic compound. Potassium ferrocyanide was used to prevent nonspecific oxidation of MCDP. The enzymatic method exhibited good analytical performance: precision, within-run CVs <1.0% and between-day CVs <2.0%; average analytical recovery, 99.3% +/- 1.8%; detection limit, 1.0 micromol/L in hemolysate; and linearity, at least up to 500 micromol/L as creatine concentration in hemolysate. Excellent agreement was observed between the present method (y) and HPLC (x), y = 1.029x - 0.002 micromol/g hemoglobin, r = 0.9998, S(y/x) = 0.053 micromol/g hemoglobin (n = 110). No significant interference was produced by various compounds, including guanidino compounds, amino acids, and reducing materials. The reference intervals (mean +/- 2 SD) for erythrocyte creatine obtained from 60 males and 60 females were (in micromol/g hemoglobin) 1.18 +/- 0.52 (0.66-1.70) for males and 1.35 +/- 0.49 (0.86-1.84) for females. Using this method, we documented changes in erythrocyte creatine in patients with various hemolytic conditions, including hemolytic anemia, liver cirrhosis, renal insufficiency, and chronic renal failure treated with hemodialysis with or without the administration of erythropoietin. We conclude that the use of MCDP allows sensitive measurement of erythrocyte creatine and that MCDP with potassium ferrocyanide can improve the sensitivity of assays that use peroxidase for detection of H2O2.     

Perioperative coagulopathy in patients undergoing primary cytoreduction

BACKGROUND: This study was performed to determine the frequency of a perioperative coagulopathy in patients undergoing primary cytoreduction for ovarian cancer or carcinoma of the peritoneum and to identify variables that might predict this phenomenon. METHODS: A retrospective review of 90 patients undergoing primary cytoreduction for ovarian cancer or carcinoma of the peritoneum was performed at Cedars Sinai Medical Center. Univariate analysis was performed to test the relationship between 15 variables and coagulopathy status. RESULTS: Six patients (6.7%) developed a perioperative coagulopathy that was unrelated to preoperative subcutaneous heparin or dilution. Coagulation disturbances developed intraoperatively before packed erythrocyte replacement equivalent to one blood volume. Four patients (4.4%) required a repeat laparotomy due to continued postoperative bleeding unresponsive to blood component replacement. Vascular pedicles were not the cause of bleeding in any patient. Univariate analysis demonstrated a significant association between perioperative coagulopathy and the following variables: ascites volume (P = 0.009), estimated blood loss (P = 0.002), preoperative serum albumin less than 3.5 g/dl (P < 0.0001), and metastasis greater than 10 cm (P = 0.033). CONCLUSIONS: Patients undergoing primary cytoreduction who have ascites, preoperative serum albumin less than 3.5 g/dl, or metastases greater than 10 cm may be at increased risk for development of a perioperative coagulopathy.     

Relationship between erythrocyte membrane permeability and the concentration in it of total cholesterol and its fractions and possible seasonal influences on these indices

A correlation between the content of labilly connected cholesterol fraction in erythrocytes and their penetration for urea was demonstrated. The dependence of erythrocyte membrane penetration on general cholesterol content and cholesterol fractions in the membrane was found to be season-influenced. A biochemical mechanism responsible for the changes in the labilly connected cholesterol fraction in erythrocyte membrane is discussed.     

Indices of membrane alterations in beta-thalassemic erythrocytes

Analytical protocols for the study of thalassemic erythrocyte membrane alterations are described. Denatured hemoglobin derivatives and aggregated band 3 are separated from detergent membrane extracts by gel-filtration as high-molecular-weight aggregates and quantitated spectrophotometrically. Membrane-bound, low-molecular-weight iron is measured on SDS-solubilized ghosts by a ferrozine-based colorimetric test. We adapted these methods for microscale preparation and analysis of erythrocyte ghosts in order to have suitable tools to estimate oxidative membrane damage in human samples. Data from 11 beta-thalassemia intermedia patients and from 10 normal controls are reported as an example of the application of these methods.     

Experimental benznidazole encephalopathy: II. Electroencephalographic and morphological alterations

Glycated hemoglobin can be degraded by proteolytic enzyme(s) in the erythrocyte. The enzyme(s) co-elutes with glycated hemoglobin when the latter is separated from erythrocyte lysates using the cation-exchanger Bio Rex-70. A further purification of the Bio Rex eluant on DEAE Sephadex A-50 separated the enzyme(s) from glycated hemoglobin. Studies with the Bio Rex eluant showed that degradation of glycated hemoglobin is maximum at 37 degrees C at pH 8.6. Proteolytic degradation is inhibited by 5 mM N-ethylmaleimide (NEM), 5 mM ethylenediamine tetraacetic acid (EDTA) and 0.6 mM n-p-tosyl-L-lysine choromethyl ketone (TLCK) (100-87 and 76% inhibition respectively). This study also examines the possibility that oxidative-damage to glycated hemoglobin increases its susceptibility to proteolytic degradation. When incubated with various anti-oxidants like DTPA, uric acid, mannitol and butylated hydroxy toluene (BHT), proteolytic degradation of glycated hemoglobin decreased by 66.1, 50.7 and 38% respectively.     

Cognitive functions of school children with normal IQ and histories of severe and early malnutrition

Reference values of some hematologic and plasma chemical parameters were established in two species of clinically normal Cercopithecidae. The following variables were studied in seven mandrills (Mandrillus sphinx) and nine white-crowned mangabeys (Cercocebus torquatus lunulatus): hematocrit, hemoglobin concentration, erythrocyte and leucocyte counts, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, glucose, urea, uric acid, cholesterol, creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine phosphokinase, lactic dehydrogenase, gamma glutamyl transpeptidase, total serum proteins, albumin, globulins, albumin-globulin ratio, sodium, potassium, calcium, magnesium, total phosphorus, chloride, and serum osmolality. Few differences were observed when compared with human hematological data and with other species of Cercopithecidae Primates.     

Amiodarone affects membrane water permeability properties of human erythrocytes and rat mitochondria

Dose-dependent water exchange times and intracellular water contents were measured by NMR (nuclear magnetic resonance) in erythrocytes and mitochondria interacted with the anti-anginal and anti-arrhytmic agent, amiodarone. Addition of the drug up to 26 microM yielded 80% enhancement of the water exchange rate in erythrocytes at 37 degrees C and 41% enhancement at 22 degrees C with 40% and 9%, respectively, increases in the intracellular water content. Similar enhancements were obtained in mitochondria at 22 degrees C. The data suggests a somewhat higher affinity of amiodarone to mitochondrial than to erythrocyte membranes.     

Phospholipid composition of the mammalian red cell membrane can be rationalized by a superlattice model

Although the phospholipid composition of the erythrocyte membrane has been studied extensively, it remains an enigma as to how the observed composition arises and is maintained. We show here that the phospholipid composition of the human erythrocyte membrane as a whole, as well as the composition of its individual leaflets, is closely predicted by a model proposing that phospholipid head groups tend to adopt regular, superlattice-like lateral distributions. The phospholipid composition of the erythrocyte membrane from most other mammalian species, as well as of the platelet plasma membrane, also agrees closely with the predictions of the superlattice model. Statistical analyses indicate that the agreement between the observed and predicted compositions is highly significant, thus suggesting that head group superlattices may indeed play a central role in the maintenance of the phospholipid composition of the erythrocyte membrane.     

Safety and immunogenicity of Brucella abortus strain RB51 vaccine in pregnant cattle

L-Asparaginase derived from Erwinia chrysanthemi which is being investigated as an alternative to E. coli for the treatment of lymphoblastic leukaemia has been found in our laboratory to lose activity upon exposure to consecutive freeze-thaw cycles. An investigation was undertaken using several techniques to characterize fully the physicochemical changes L-Asparaginase is undergoing during freeze-thaw cycling leading to the loss of its activity. A total protein assay suggested that the loss of some enzyme activity was a result of protein precipitation. Circular dichroism (CD) studies showed a decrease of alpha-helical structure with a concomitant increase in beta sheet and random coil content, suggesting alterations in the secondary structure leading to unfolding, the first step of denaturation processes. The elution profiles obtained from size-exclusion chromatography (SEC) studies indicated the formation of several species during the process of freezing and thawing. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) studies showed bands corresponding to 1-3 kDa and 32 kDa, suggesting that some of the species are fragments and shortened monomers resulting from cleavage of monomers. The molecular weight distribution obtained using SEC-linked light scattering indicated a substantial fraction of polydispersed fragments ranging from 900 Da to 3 kDa and a small fraction of aggregates corresponding to 300 kDa. A scheme was proposed to explain the cascade of events leading to the loss of soluble protein and accompanying loss of enzyme activity. Tetramers of the enzyme dissociate into monomers some of which are cleaved into small fragments. The shortened monomers then aggregate and precipitate.     

Partial recovery of erythrocyte glycogen in diabetic rats treated with phenobarbital

Erythrocytes may play a role in glucose homeostasis during the postprandial period. Erythrocytes from diabetic patients are defective in glucose transport and metabolism, functions that may affect glycogen storage. Phenobarbital, a hepatic enzyme inducer, has been used in the treatment of patients with non-insulin-dependent diabetes mellitus (NIDDM), increasing the insulin-mediated glucose disposal. We studied the effects of phenobarbital treatment in vivo on glycemia and erythrocyte glycogen content in control and alloxan-diabetic rats during the postprandial period. In control rats (blood glucose, 73 to 111 mg/dl in femoral and suprahepatic veins) the erythrocyte glycogen content was 45.4 +/- 1.1 and 39.1 +/- 0.8 micrograms/g Hb (mean +/- SEM, N = 4-6) in the femoral artery and vein, respectively, and 37.9 +/- 1.1 in the portal vein and 47.5 +/- 0.9 in the suprahepatic vein. Diabetic rats (blood glucose, 300-350 mg/dl) presented low (P < 0.05) erythrocyte glycogen content, i.e., 9.6 +/- 0.1 and 7.1 +/- 0.7 micrograms/g Hb in the femoral artery and vein, respectively, and 10.0 +/- 0.7 and 10.7 +/- 0.5 in the portal and suprahepatic veins, respectively. After 10 days of treatment, phenobarbital (0.5 mg/ml in the drinking water) did not change blood glucose or erythrocyte glycogen content in control rats. In diabetic rats, however, it lowered (P < 0.05) blood glucose in the femoral artery (from 305 +/- 18 to 204 +/- 45 mg/dl) and femoral vein (from 300 +/- 11 to 174 +/- 48 mg/dl) and suprahepatic vein (from 350 +/- 10 to 174 +/- 42 mg/ dl), but the reduction was not sufficient for complete recovery. Phenobarbital also stimulated the glycogen synthesis, leading to a partial recovery of glycogen stores in erythrocytes. In treated rats, erythrocyte glycogen content increased to 20.7 +/- 3.8 micrograms/g Hb in the femoral artery and 30.9 +/- 0.9 micrograms/g Hb in the suprahepatic vein (P < 0.05). These data indicate that phenobarbital activated some of the insulin-stimulated glucose metabolism steps which were depressed in diabetic erythrocytes, supporting the view that erythrocytes participate in glucose homeostasis.