Corticosteroids do not alter the threshold for vertebral fracture.

Corticosteroid use is one of the most important secondary causes of osteoporosis. Generally, it has been believed that in addition to its effect on bone mineral density (BMD), it also causes an alteration in bone quality that means that fractures occur at a lower BMD than might be expected. To establish if this is the case, we have compared the relationship between BMD and vertebral fracture in patients receiving corticosteroids with that in patients who had never received such therapy. Information was gathered on those patients who had been referred to the participating centers and had both BMD measurements and lateral thoracolumbar radiographs. In all, 452 patients (391 female) were identified; of these 82 (63 female) were receiving corticosteroids. There was no significant difference in BMD between the patients on corticosteroids and those with other suspected causes of osteoporosis. Vertebral fractures were present in 53% of patients on steroids compared with 35% of those who had no such treatment (p = 0.0035). The fractures were more likely to be multiple in patients on corticosteroids (p = 0.0042). However, if the relationship between bone density and fracture is investigated by plotting the cumulative prevalence of fracture against the bone density, measured by T score, the median BMD for fractures actually was marginally lower in patients on steroids, -2.74 (95% confidence interval [CI], -2.77 to -2.70) compared with -2.65 (95% CI, -2.66 to -2.65) in those who had not received steroids. Our results fail to support the notion that the fracture threshold is altered in patients on long-term steroids and suggest that the same diagnostic criteria should be used for osteoporosis in patients whether or not they are taking corticosteroid therapy.

Asbestos contamination in paraffin tissue blocks.

Electron microscopic analyses of tissue samples embedded in paraffin are routinely performed as a means of validating (establishing) the asbestos exposure of persons diagnosed with mesothelioma, a rare form of cancer often identified as being caused by exposure to asbestos. During analysis of tissue samples claimed to contain asbestos, we observed asbestos contamination in the paraffin of the tissue block. To investigate the extent to which such contamination is prevalent in tissue block samples, we obtained and analyzed samples of paraffin blocks from hospitals in six major cities and found them to contain measurable concentrations of asbestos. Whether this asbestos contamination originated in the virgin wax or was a result of processing has not been established. This result, which has not been previously reported, raises significant concerns about the validity of analyses for asbestos in tissue embedded in paraffin. In particular, diagnoses in which the presence of asbestos in tissue samples is taken as being indicative of past asbestos exposure, especially for those cases in which no known exposure has occurred, and studies purporting to show migration of asbestos to other organs in the body following inhalation or ingestion of asbestos require critical reevaluation. The need for reevaluation is particularly acute if appropriate control blanks were not evaluated as part of the studies.

In vitro characteristics of 'undercollected' units of whole blood in CP2D-A.

Blood donation volumes less than 350 ml are classified as 'undercollected' at the NSW Red Cross Blood Transfusion Service (BTS) and are discarded. This study evaluated the in vitro characteristics during storage of both undercollected units and units of acceptable volume. Thirty-two units of whole blood were each collected into 63 ml of CP2D-A, with blood volumes ranging from 180 to 456 ml. The units were stored between 4 and 6 degrees C for 35 days and in vitro measurements were performed weekly. Biochemical parameters measured included ATP, extracellular pH, total haemoglobin, haematocrit, mean cell volume, plasma sodium and potassium, plasma haemoglobin, 2,3-DPG and lactate levels. All parameters were within the BTS acceptable quality control limits for whole blood. Thus, it appears feasible to transfuse undercollected units with volumes between 180 and 350 ml. However, routine transfusion of undercollected homologous units is undesirable. In contrast, it may be preferable to transfuse an autologous unit, even if it was undercollected. The performance of in vivo survival studies would provide confirmatory data on this proposition.

Red cells suspended in a phosphate-fortified additive: biochemical enhancement and longer storage.

When compared to our current storage regime, an additive solution, with 75 mM inorganic phosphate, improved certain biochemical and morphological parameters measured in suspensions of packed red cells throughout 49 days of liquid storage. In particular, the mean ATP level of 20 donations stored with the high phosphate additive was consistently and significantly higher than in standard (n = 6) suspensions. The increased amount of inorganic phosphate and the higher pH of the new additive stimulated ATP production and provided better pH buffering than the standard solution currently in use. Although survival study results indicated adequate 24h survivals for erythrocytes stored for 42 days with the new solution, after 49 days storage, the mean 24h survival of autologous erythrocytes was only 58 +/- 7% (n = 6).

31P magnetization transfer in the phosphoglyceromutase-enolase coupled enzyme system.

The rate of exchange of phosphoryl groups between 2-phosphoglycerate, 3-phosphoglycerate and phosphoenolpyruvate by the coupled phosphoglyceromutase-enolase enzyme system using one- and two-dimensional 31P NMR spectroscopy was measured. Magnetization exchange in one-dimensional experiments was achieved by saturation transfer with selective irradiation at both one and two sites in this three-site exchange system using the DANTE pulse sequence. The two-dimensional magnetization exchange experiment avoids the need to selectively saturate at one or more frequencies which may be difficult in complex exchange systems. Analysis of the two-dimensional exchange experiment by the back transformation method yielded exchange rate constants in good agreement with the saturation transfer method.

Supernatant hemoglobin determinations after prolonged blood storage.

Measurement of extracellular hemoglobin is useful for assessing the relative success of different blood storage strategies. The test is now also included in routine quality assurance procedures. With the recent trend towards transfusion of concentrated red cells resuspended in "additive" solutions, a safe and efficient method for the estimation of hemoglobin in these units is required. We have developed a method suitable for this purpose, based on the formation of cyanomethemoglobin. Tetramethylbenzidine (TMB) methods were also investigated and shown to detect hemoglobin and hemoglobin derivatives quantitatively when present in packed cell supernatants. A close correlation was recorded between the estimates of supernatant hemoglobin using the 2 techniques with 68 stored red cell concentrates. Because of its simplicity, adequate sensitivity and avoidance of carcinogenic benzidine derivatives, we recommend the cyanomethemoglobin method for routine use in the measurement of supernatant hemoglobin in stored modified red cell concentrates.

Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure.

Methylphosphonate in conjunction with 31P-NMR spectroscopy was used for the measurement of transmembrane delta pH in human erythrocytes stored at 4 degrees C for up to 5 weeks in a nutrient medium. Intra- and extracellular pH was determined using calibration curves based on the pH-dependent separation between the NMR resonances of methylphosphonate and orthophosphate (Pi). A comprehensive statistical procedure is presented for the determination of the variance of NMR-based pH estimates. The entry of methylphosphonate into erythrocytes was more rapid at low pH and uptake was fully inhibited by the band 3 reagent, disodium 4,4-diisothiocyano-2,2'-disulphonic acid stilbene. The distribution ratio of methylphosphonate concentration inside and outside the cells was used to calculate the membrane potential; the analysis depends on a consideration of the Donnan equilibrium for an anion with one or two charges. Furthermore, the analysis does not depend on the pH estimates but relies solely on concentration estimates. The chemical shift of methylphosphonate was not subject to the variations associated with specific intracellular binding encountered with many other phosphorus compounds, including Pi. On the other hand, the ionic strength dependence of the chemical shift of methylphosphonate, contrary to earlier reports, is comparable in magnitude (but opposite in sign) to that of Pi.

Intra- and extraerythrocyte pH at 37 degrees C and during long term storage at 4 degrees C: 31P NMR measurements and an electrochemical model of the system.

A 31P NMR method based on pH dependent variation of the chemical-shift-difference between the resonances of orthophosphate and methylphosphonate was used to measure simultaneously intracellular pH (pHi) and extracellular pH (pHc) during long term storage of erythrocytes; pH was determined at both 4 degrees C and 37 degrees C. An equation describing the equilibrium distribution of membrane-permeant ions was derived by consideration of the electrochemical and osmotic constraints in the RBC suspension. Calculations using the model-equation and the measured pHi yielded the Donnan ratio and therefore pHo; the relationship between experimentally determined pHi and pHo values was accurately predicted by the model. Sensitivity analysis of the model-equation revealed that the observed increase in transmembrane pH gradient during storage is principally due to the alteration of the total net charge of intracellular (poly)-anions.

N2-(1-carboxyethyl)methionine. A 'pseudo-opine' in octopine-type crown-gall tumours.

A novel methionine-containing plasmid-determined compound, N2-(1-carboxyethyl)methionine (NCEM) has been identified in crown-gall tumours induced by octopine-type strains of Agrobacterium tumefaciens. NCEM is probably synthesized by octopine synthase. Cell-free preparations from octopine-type strains of A. tumefaciens can degrade NCEM; however, the bacterium cannot transport the compound into the cell, although these strains can take up and degrade the octopine family of opines.

Regeneration of phosphorylated metabolites in stored erythrocytes in an open perfusion system: studies using 31P NMR spectroscopy.

Human erythrocytes were maintained at high haematocrit in a metabolically functional state for several hours in a thermodynamically open perfusion apparatus. The concentrations of ATP and 2,3-bisphosphoglycerate (2,3-DPG) and pH were continuously monitored before and after metabolic perturbations by using 31P NMR; the monitoring was achieved with a 31P flow-through probe. Methylphosphonate was added to plasma perfusion medium as a phosphorus concentration standard and as a 31P NMR pH probe molecule. The rates of decline of ATP and 2,3-DPG levels in fresh cells in a glucose-free medium were measured as were the rates of reformation in response to a 'rejuvenation' medium. Also, rates of ATP and 2,3-DPG synthesis during perfusion with Krebs bicarbonate-0.5 mmol/l glucose and perfusion with pooled plasma were measured in cells that had been previously stored at 4 degrees C for 5 weeks.

Rubella: immunity and vaccination in schoolgirls.

Of 191 schoolgirls, 128 volunteered to take part in a feasibility study of serotesting before and after rubella vaccination, and all responded to RA 27/3 vaccine. Had the serum samples been taken by a fingerprick method the number of volunteers would probably have increased considerably. A change in policy for rubella vaccination to testing both before and after vaccination would cost no more than the existing policy, would ensure primary response, and would differentiate those women who were protected by the vaccine from those with antibody to wild virus.

Insulin increases body fat despite control of food intake and physical activity.

The effect of experimentally induced hyperinsulinemia on body composition was studied in rats with food intakes precisely controlled by intragastric feeding and physical activity manipulated by sedation with chlordiazepoxide (CDP). Male Sprague-Dawley rats (n = 38) were fitted surgically with gastrostomy tubes. After 8 days the animals were divided into four groups. Group 1 received daily injections of protamine-zinc insulin; group 2 received daily injections of saline; group 3 received the same insulin doses as group 1 plus daily administration of CDP mixed with the diet; group 4 received daily injections of saline plus CDP in the diet. All groups were tubefed identical amounts of semiliquid diet via gastrostomy. Physical activity was measured by electronic monitor. After 4 wk the rats were killed. The insulin-treated groups (1 and 3) had significantly larger fat depots and larger mean fat cell size than the noninsulin-treated groups (2 and 4). This increase in fat occurred concurrently with a decrease in carcass protein and water. Physical activity, as measured, was unaltered by insulin but was significantly reduced by CDP. Treatment with CDP only increased the dorsal fat depot and liver weight but had no significant effect on total dissected fat depots and had a reductive effect on carcass protein. In conclusion insulin treatment enhanced the efficiency of conversion of energy intake into fat energy stores.

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system.

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 mumol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [14C]pyruvate to haemoglobin and the rate of efflux of [14C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent Km and Vmax for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.

Intestinal glycosidase activities in one adult and two suckling echidnas: absence of a neutral lactase (beta-D-galactosidase).

The activities of various glycosidases in homogenates of the small-intestinal mucosa of one adult and two suckling echidnas, Tachyglossus aculeatus, were investigated. The activities of lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, neuraminidase and alpha-L-fucosidase were higher in the sucklings than in the adult animal. Maltase and isomaltase activities were lower. Sucrase and cellobiase activities were absent or present in trace amounts only. The lactase activity had a pH optimum of 4.0-4.5, was predominantly in the soluble fraction following ultracentrifugation and was inhibited by p-chloromercuribenzene sulfonate, suggesting that it was due to a lysosomal acid beta-galactosidase and not a brush-border neutral lactase. The maltase activity of the sucklings also had the characteristics predominantly of a lysosomal acid hydrolase. It is proposed that in suckling echidnas, the oligosaccharides (mainly neuraminyllactose and fucosyllactose) of the mother's milk are digested intracellularly by lysosomal enzymes, rather than at the brush border, of the epithelial cells of the small-intestinal mucosa.

Influence of previous gold toxicity on subsequent development of penicillamine toxicity.

The incidence of penicillamine toxicity was determined in 250 patients who had never previously received gold, 76 patients who had received gold without toxic reaction, and 79 patients with a previous history of gold toxicity. The results suggest that there may be a higher incidence of penicillamine toxicity in patients who have previously shown toxic reactions. The interval between stopping the gold and starting the penicillamine did not influence incidence of toxicity. The development of a rash during gold treatment does not seem to influence the development of a rash during penicillamine treatment, but patients who have had proteinuria or bone-marrow depression during gold treatment may have an increased likelihood of developing a similar side effect with penicillamine.