Microcalorimetric measurements of heat production in human erythrocytes. Heat effect during methylene blue stimulation.

The metabolic activity in human erythrocytes during stimulation with 10(-4) mol/l methylene blue has been studied by a microcalorimetric method. Simultaneous measurements were performed on cells from the same preparation suspended in different media. Mean values for the ratios between heat effect values were 7.1 +/- 1.0, 7.4 +/- 0.8, and 10.2 +/- 1.7 (+/- S.D.) for cells suspended in plasma, serum, and glucose phosphate buffer, respectively. All heat effect values were corrected to pH 7.40 using the correction factor found in the present work (0.4 % per 0.01 pH unit). The present calorimetric results are in qualitative agreement with previous reports of other investigators concerning the stimulating effect of methylene blue and the influence of pH on the pentose phosphate pathway.     

Microcalorimetric measurements of heat production in whole blood and blood cells of normal persons.

Microcalorimetric measurements have been made of the heat production in whole blood and its major cell fractions. All measurements were made with samples from normal subjects. The average heat effect value found (plus or minus S.D.) for whole blood was 62 plus or minus 7 mW/l. The value obtained for erythrocytes was 82 plus or minus 6 mW per liter of packed cells suspended in phosphate buffer at pH 7.4. For lymphocytes and for polymorphonuclear leukocytes heat effect values were 4.6 plus or minus 1.8 and 1.2 plus or minus 0.4 pW/cell, respectively, for cells suspended in buffer. For plasma suspensions corresponding values were 2.2 plus or minus 1.4 and 3.5 plus or minus 1.0 pW/cell, respectively. For thrombocytes suspended in plasma the heat effect value was 59 plus or minus 8 fW/cell. Heat production in cell-free plasma was close to zero. Using heat effect values determined for the different cell fractions, values could be calculated for whole blood samples which were in full agreement with the values obtained by direct measurements of whole blood.     

Stability of human blood serum aminoacids after storage at different pH and temperature conditions

The stability of the aminoacids in human blood serum ultrafiltrates and in an aminoacid standard solution was investigated under different pH and storage conditions. At close to neutral pH values the aminoacid concentrations remained constant for at least 12 months at -50 degrees C (n = 6), except for lysine. With acid but particularly with alkaline conditions a time- and temperature-dependent decrease was observed for glutamine and asparagine with concomitant increases of glutamate and aspartate. Similar, but less prominent alterations were noted for the concentrations of methionine, glycine, tyrosine, histidine, arginine and ornithine. Almost independent of pH, there was an effect of temperature; after 24 h at 55 degrees C a significant increase of several per cent in a number of serum aminoacid concentrations was observed, presumably due to the hydrolysis of small proteins and peptides. For the purpose of aminoacid analysis it is recommended that samples be stored deproteinized, deep-frozen and at neutral pH.     

The heart production in energy-depleted human erythrocytes induced by glucose, inosine and adenine.

The heat production (HP) of glucose deprived human red blood cells was measured, using glucose, adenine and inosine as substrates. Inosine induced a significantly higher HP than glucose and adenine induced no significant HP. At low pH the HP of glucose decreased more than that of inosine, corresponding to an equally lowered lactate production. The results indicate that it should be possible to use the system developed to study the functional state both of the complete glycolytic system and the lower part of it in intact red blood cells during various clinical conditions.     

Influence of time and storage conditions on plasma HIV viral load measurements

Quantification of human immunodeficiency virus (HIV) RNA in plasma from HIV-infected patients is now widely used as a clinical indicator of disease prognosis and of response to antiretroviral therapy. However, controversy exists as to whether values obtained under different testing conditions could vary significantly, thus jeopardizing the appropriate interpretation of data. Herein, we demonstrate that results obtained after testing plasma versus whole blood, or immediate versus deferred processing, do not appear to influence viral load measurements significantly. Thirty blood samples from HIV-infected patients were analysed. The second generation branched-DNA assay was used for quantification of plasma viral load. HIV RNA remained stable for at least 24 h at room temperature, either in plasma or in whole blood, in 72.4% of the samples (< 0.2 log difference in viral load values) although lower levels of HIV RNA tend to be seen in samples after being stored as whole blood at room temperature. Only 3.4% of samples showed a decline > 0.5 log when they were left as whole blood at room temperature for 24 h in comparison with testing after immediate plasma separation. Although immediate separation and refrigeration of plasma samples may reduce the chance of significant falls in viral load measurements, this level of processing can be limited in regions where clinical blood samples cannot be processed rapidly. Our data provide confidence in the results obtained when testing specimens, either plasma or whole blood, 24 h after venepuncture and storage at room temperature, mimicking the conditions in the transport of blood samples to reference centres.     

Oxypurine cycle in human erythrocytes regulated by pH, inorganic phosphate, and oxygen.

The effect of pH, PO2, and inorganic phosphate on the uptake and metabolism of hypoxanthine by erythrocytes has been studied. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low PO2. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favoring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate. Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3-bisphosphoglycerate. These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely 5'-phosphoribosyl-1-pyrophosphate synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue.     

Relationship between plasma glucose and insulin concentration, glucose production, and glucose disposal in normal subjects and patients with non-insulin-dependent diabetes.

The changes in hepatic glucose production (Ra), tissue glucose disposal (Rd), and plasma glucose and insulin concentration that took place over a 16-h period from 10 to 2 p.m. were documented in 14 individuals; 8 with non-insulin-dependent diabetes mellitus (NIDDM) and 6 with normal glucose tolerance. Values for Ra were higher than normal in patients with NIDDM at 10 p.m. (4.73 +/- 0.41 vs. 3.51 +/- 0.36 mg/kg per min, P less than 0.001), but fell at a much faster rate throughout the night than that seen in normal subjects. As a consequence, the difference between Ra in normal individuals and patients with NIDDM progressively narrowed, and by 2 p.m., had ceased to exist (1.75 +/- 0.61 vs. 1.67 +/- 0.47 mg/kg per min, P = NS). Plasma glucose concentration also declined in patients with NIDDM over the same period of time, but they remained quite hyperglycemic, and the value of 245 +/- 27 mg/dl at 2 p.m. was about three times greater than in normal individuals. Plasma insulin concentrations also fell progressively from 10 to 2 p.m., and were similar in both groups throughout most of the 16-h study period. Thus, the progressive decline in Ra in patients with NIDDM occurred despite concomitant falls in both plasma glucose and insulin concentration. Glucose disposal rates also fell progressively in both groups, but the magnitude of the fall was greater in patients with NIDDM. Consequently, Rd in patients with NIDDM was higher at 10 p.m. (3.97 +/- 0.48 vs. 3.25 +/- 0.13 mg/kg per min, P less than 0.001) and lower the following day at 2 p.m. (1.64 +/- 0.21 vs. 1.97 +/- 0.35 mg/kg per min, P less than 0.01). These results indicate that a greatly expanded pool size can exist in patients with NIDDM at a time when values for Ra are identical to those in normal subjects studied under comparable conditions, which suggests that fasting hyperglycemia in NIDDM is not simply a function of an increase in Ra.     

31P NMR measurements of intracellular pH and 2,3-diphosphoglycerate in erythrocytes

31P nuclear magnetic resonance (NMR) spectroscopic determinations of 2,3-diphosphoglycerate (2,3-DPG) concentrations in erythrocytes have been compared with values obtained by an enzymatic assay. It was found that the mean difference over a concentration range from 2 to 20 mmol 2,3-DPG/1 red blood cells (RBC) was 0.18 +/- 0.39 (SEM) mmol/l RBC (n = 10). 31P NMR spectroscopy was further tested by simultaneous measurements of 2,3-DPG and intracellular pH under conditions where the concentration of 2,3-DPG was varied. Thereby a linear relation between the pH gradient (delta pH) over the erythrocyte membrane and 2,3-DPG concentrations was found. The data fit a Donnan equilibrium model for the pH difference over the erythrocyte membrane. We conclude that 31P NMR spectroscopy is a sensitive and accurate method that makes possible simultaneous and non-invasive measurements of intracellular pH and 2,3-DPG.     

The effect of time and temperature on blood glucose measurements.

Diabetic patients admitted for home healthcare services may require daily blood glucose measurements by the nurse until glucose control is achieved. Improper storage of blood specimens and unstable environmental temperature may alter the reliability of blood glucose measurements. Because accurate measurements are essential in decision making for diabetic control, specimen collection and handling are important concerns for home healthcare nurses.     

The effect of storage conditions on lipid peroxidation in erythrocytes

Lipid peroxidation products in red cells should be examined within 24 hrs after blood collection. If it is impossible, the blood or treated red cells can be stored at 4-6 degrees C for up to 3 days. A lower storage temperature promotes a drastic reduction of diene conjugate and malonic dialdehyde levels within the first 24 hrs.     

Regulation of lactate uptake and lactate production in liver from 48-h-starved rats: effects of pH, flow and glucose concentration

1. The effects of medium glucose concentration (0-20 mmol/l) and flow (100-33% of normal) on lactate uptake at low lactate concentration were studied in perfused livers from 48-h-starved rats with perfusate pH values of 7.4 and 6.8. 2. Lactate uptake was independent of glucose concentration in the range 5-10 mmol/l, but was slightly inhibited with time at 20 mmol/l glucose. This pattern was independent of perfusate pH. 3. At both pH values lactate uptake decreased proportionally with flow, and at low flow lactate was produced by the livers. The effect of flow was greatest at pH 7.4 where a net lactate production was found at 48% of normal flow, whereas at pH 6.8 lactate production was not seen until the flow was reduced to 33% of normal. 4. When glucose was omitted from the perfusate lactate production ceased at both pH values. 5. The effect of low pH on lactate uptake and production in liver probably reflects inhibition of glycolysis by low pH.     

Energy metabolism in human erythrocytes: II. Effects of glucose depletion

Normal red cells were incubated in the absence of glucose to develop a system in which total adenosine triphosphate (ATP) turnover could be assessed. After 1 hr, the triose pool had been completely consumed. Thereafter, the metabolism of 2,3-diphosphoglycerate (DPG) to pyruvate and lactate was the sole significant source of ATP synthesis.10(-3)M CuCl(2), which did not enter the cells, diminished ATP utilization by more than 50%. This could be only partially attributed to the inhibition by copper of residual acylation and cation pumping, which were already reduced by glucose depletion. Other membrane enzymes, which presumably function in the maintenance of membrane integrity, must, therefore, use a significant portion of erythrocyte ATP.The behavior of glucose-depleted red cells with respect to cation transport was complex. The addition of ouabain did not decrease ATP utilization in these red cells. Ouabain inhibitable potassium influx was nearly normal after triose depletion, but total potassium influx was decreased. In contrast, the ouabain inhibitable sodium efflux was markedly reduced after triose depletion, although the concentration of ATP was 70% of normal. The dissociation of monovalent cation pumping suggests that the energy for active sodium transport is derived from a specific source (such as the ATP produced by the phosphoglycerate kinase reaction) distinct from that for potassium transport.     

Determination of porphobilinogen deaminase activity in human erythrocytes: pertinent factors in obtaining optimal conditions for measurements

Determination of porphobilinogen deaminase (PBGD; EC 4.3.1.8) activity in erythrocytes can contribute to the identification of patients suspected of acute intermittent porphyria. PBGD catalyses the polymerization of four molecules of porphobilinogen (PBG) to the highly unstable 1-hydroxymethylbilane. The 1-hydroxymethylbilane is transformed into uroporphyrinogen III by uroporphyrinogen III synthase. When this enzyme is inactivated, 1-hydroxymethylbilane cyclizes non-enzymatically to uroporphyrinogen I, which can be oxidized to uroporphyrin I. PBGD activity can be measured by quantitation of uroporphyrin I formed from PBG under conditions where this is the only end product. The purpose of the present study was to define the optimal conditions for quantitating PBGD activity in human erythrocytes. The preanalytical factors examined were: anticoagulants and methods for disruption of the erythrocytes. The analytical factors examined were: duration of preincubation, reaction time, reaction temperature, pH, ionic strength and conditions for the oxidation of uroporphyrinogen I to uroporphyrin I. Based on the results, we propose an optimized method for determination of PBGD activity in erythrocytes.     

Effects of erythrocytes, bicarbonate, temperature and albumin on in vitro ionized calcium variations with pH

Using a heart lung machine as an in vitro model, the relation between ionized calcium (cCa2+) and pH has been shown to depend on several variables such as erythrocytes, temperature and albumin as well as the total calcium and bicarbonate concentrations. The respiratory acid-base disturbances were simulated by changing the gas flow between 1.0 and 2.41 min-1 by adding CO2, to the machine at a concentration of 0 to 17%. When pCO2 was used to alter pH, cCa2+ varied from 0.16 mmol l-1 per pH unit to 0.52 mmol l-1. The regression slope of cCa2+ on pH was made steeper by decreasing erythrocyte volume fraction and by increasing temperature and the concentrations of HCO3, calcium or albumin. The metabolic acid-base alterations were produced by HCl or NaHCO3 at a constant gas flow, cCA2+ changes per pH unit were 0.70 mmol l-1 in plasma and 1.04 mmol l-1 in whole blood. The different results found in plasma and in erythrocyte fluids may be explained by their different buffering capacity. Haemoglobin may buffer hydrogen ions, and the formation of HCO3- is catalysed by carbonic anhydrase from the red cells.     

Determination of porphobilinogen deaminase activity in human erythrocytes: pertinent factors in obtaining optimal conditions for measurements

Determination of porphobilinogen deaminase (PBGD; EC 4.3.1.8) activity in erythrocytes can contribute to the identification of patients suspected of acute intermittent porphyria. PBGD catalyses the polymerization of four molecules of porphobilinogen (PBG) to the highly unstable 1-hydroxymethylbilane. The 1-hydroxymethylbilane is transformed into uroporphyrinogen III by uroporphyrinogen III synthase. When this enzyme is inactivated, 1-hydroxymethylbilane cyclizes non-enzymatically to uroporphyrinogen I, which can be oxidized to uroporphyrin I. PBGD activity can be measured by quantitation of uroporphyrin I formed from PBG under conditions where this is the only end product. The purpose of the present study was to define the optimal conditions for quantitating PBGD activity in human erythrocytes. The preanalytical factors examined were: anticoagulants and methods for disruption of the erythrocytes. The analytical factors examined were: duration of preincubation, reaction time, reaction temperature, pH, ionic strength and conditions for the oxidation of uroporphyrinogen I to uroporphyrin I. Based on the results, we propose an optimized method for determination of PBGD activity in erythrocytes.