The origin of plasma creatine kinase 1, detected in patients during and following open heart surgery.

Plasma creatine kinase 1 (CK-1) was detected intra-operatively in 6 out of 6 patients and postoperatively in 15 out of 22 patients, undergoing cardiac surgery. A transient increase in plasma levels of creatine kinase 2 (CK-2) and total creatine kinase (CK-tot.) activity was observed in all patients. The disappearance rates for the 2 isoenzymes in the circulation were CK-1: Kd = 4.7 X 10(-3) min-1, and CK-2: Kd = 0.60 X 10(-1) min-3. Analysis of vessel and heart tissue showed that the saphenous vein contained mainly CK-1; high activities of all three isoenzymes were found in the parts of the heart investigated. Most probably, both CK-1 and CK-2 are liberated from injured cardiac tissue.

A new physiological biomarker for nitrate exposure in humans.

After toxicological studies with nitrate/nitrite in rats it was observed with nuclear magnetic resonance that N-methylnicotinamide (NMN), a metabolite of tryptophan was increased. The use of NMN as a biomarker for nitrate/nitrite exposure was investigated further in additional experiments with rats and in a human study with volunteers. Rats have been exposed to 36 mmol KCl, KNO2 or KNO3 per 1 tap water for 13 weeks. In general, the animals receiving KNO2 showed a statistically significant (P < 0.01) 2-fold increase in NMN compared with the KCl group. This increase was observed after a relatively high exposure (about 800 mg/kg body wt./day). It was also noticed that the initial increase in urinary NMN concentrations decreased after prolonged exposure for 12 weeks. To investigate the induction of urinary NMN in humans, an experiment has been performed in which 8 volunteers received a single oral dose of sodium nitrate, corresponding with 10 mg NaNO3/kg body wt./day (2 times the acceptable daily intake for nitrate). A rapid increase of urinary NMN (up to 6-fold) was observed in 4 volunteers. In the other 4 volunteers the urinary NMN concentration did hardly react. When the experiment was repeated with the same volunteers, it was remarkable to see that in this experiment all volunteers showed the same individual response on urinary NMN as in the first experiment. It is concluded that NMN can possibly be a good biomarker for the internal nitrite exposure of humans, but further studies are necessary to assess its value.

Effect of nitrite on blood pressure in anaesthetized and free-moving rats.

The effect of nitrite on blood pressure and heart rate was studied in anaesthetized (non-telemetric method) and free-moving rats (biotelemetry system). In anaesthetized rats, NaNO2 (10-1000 mumol/kg), infused over 5 min, induced a dose-related decrease in blood pressure. The maximal decrease in mean arterial blood pressure (MAP), caused by 1000 mumol/kg NaNO2 and measured 15 min after infusion was 55.9 +/- 3.2% (n = 3). After NaNO2 infusion, in the plasma, rapid conversion of nitrite into nitrate was observed. However, sodium nitrate (NaNO3, 100 mumol/kg) did not decrease blood pressure and there was no conversion of nitrate into nitrite. Free-moving rats received KNO2 which was added to drinking water (36 mmol/litre) for a period of 3 days. KNO2 decreased the MAP and increased the heart rate during the rat's activity phase at night but not during their resting phase in the day. An equal concentration of potassium (KCl, 36 mmol/litre added to drinking water) for 3 days did not decrease blood pressure. It is concluded that nitrite decreases blood pressure in rats, which probably induces, by renin-angiotensin system activation, hypertrophy of the adrenal zona glomerulosa.

Biochemical and histological alterations in rats after acute nitrogen dioxide intoxication.

1. In previous studies a rat inhalation model was developed to investigate the treatment of acute nitrogen dioxide (NO2) intoxication. 2. Biochemical parameters, which may be important for the evaluation of lung injury and repair, were reviewed and compared with the histology. 3. After exposure to high NO2 concentrations (75 ppm, 125 ppm or 175 for 10 min) the lung injury observed by light microscope was most pronounced after 24 h and became worse with increasing concentration. 4. The most sensitive indicators for lung injury in the broncho-alveolar lavage fluid (BAL) were protein and albumin concentrations, angiotensin converting enzyme activity, beta-glucuronidase activity and the presence of neutrophil leucocytes. The changes observed in these variables were dose-dependent. Following exposure to 175 ppm the protein and albumin concentrations and the angiotensin converting enzyme activity showed a 100-fold increase, while the beta-glucuronidase activity showed a 10-fold increase. 5. Glucose-6-phosphate dehydrogenase and glutathione peroxidase in the supernatant of lung homogenate and gamma-glutamyl transferase activity in BAL are likely to be the most practical parameters for monitoring the phase of repair because their activities were maximal at the moment histological changes were reduced in intensity. 6. Repair was almost complete 7 d following exposure.

The mechanism of fluoride-induced hypocalcaemia.

1. Fluoride intoxication leads to sudden cardiac death which has been assumed to result from the accompanying severe hypocalcaemia. The aim of this study has been to investigate the suggestion that fluorapatite formation rather than CaF2 precipitation is responsible for this low calcium. 2. Measurements of free Ca2+ and F- ion concentrations in HEPES buffered solutions containing F-, Ca2+, and phosphate ions at different concentrations in the absence and presence of hydroxyapatite showed that the presence of hydroxyapatite enhanced the decrease of Ca2+ and F- concentration. 3. The ratio of Ca2+:F- clearance was 5:1 which is consistent with formation of fluorapatite. These results support the hypothesis that hydroxyapatite acts as a nucleation catalyst for fluorapatite formation and this process is responsible for the hypocalcaemia induced by fluoride intoxication. 4. The proposed mechanism explains also the metabolic acidosis which is frequently seen in cases of fluoride intoxication.

Development of a rabbit model to investigate the effects of acute nitrogen dioxide intoxication.

1. In previous studies a rat inhalation model was developed to investigate the effects of intervention after acute NO2 exposure. The object of the present study was to investigate whether acute NO2 intoxication induced comparable effects in rabbits as it does in rats. Where the effects of intervention in both species are similar, then the conclusions drawn from these studies may have more relevance for the treatment of man. 2. Biochemical variables in bronchoalveolar lavage and supernatant from lung homogenate, which may be relevant for the evaluation of lung injury and repair, were investigated and compared with histology. 3. After NO2 exposure for 10 min, the pulmonary effects observed became more pronounced with increasing NO2 concentrations (0, 125, 175, 250, 400, 600 or 800 ppm) [1 ppm NO2 is 1.88 mg m-3]. The effects in rabbits were found to be broadly comparable with those in rats. 4. To achieve severe lung injury in rabbits without mortality, enabling investigations of the effects of intervention over several days, exposure to a NO2 concentration of 600 ppm for 10 min was most appropriate, while a concentration of 175 ppm NO2 was needed to attain comparable effects in rats. 5. The repair phase starts later, namely at 3 days after exposure in rats, compared to 5 days in rabbits.

Nasal lavage as tool for health effect assessment of photochemical air pollution.

It is widely accepted that humans exposed to known concentrations of ozone under controlled conditions exhibit reversible changes that affect the large and small airways as well as the alveolar region of the lung. Among the reversible changes, the induction of inflammatory responses in the lung are of major concern. Many of the cell types found in the lining of the nasopharyngeal region are similar to cells of the tracheal and bronchial lining. therefore, it has been suggested that the cellular responses in the nose to toxicants are likely to be similar to the lower airway at the same dose of the agent. If these pollutants are respiratory irritants, capable of causing cellular damage, effects may therefore be detected in the nasal passage. Experimental studies have shown that the inflammatory response in the nose may be predictive for the situation in the lung. In this paper we described the results of a feasibility study on the use of nasal lavage for epidemiological studies. Nasal lavages were performed in 12 volunteers, 5-7 times per volunteer during 2 months. Polymorph nuclear leukocytes (PMN's), immune mediators and markers for exudation were monitored in the nasal lavage (NAL). It was found that the procedure of the nasal lavage technique was minimally invasive, very well tolerated and no adverse side effect were observed. The leukocytes, the proteins myeloperoxidase (MPO), eosinophil cationic proteins myeloperoxidase (MPO), eosinophil cationic protein (ECP) and interleukin-8 (IL-8) were detectable in NAL of most volunteers, while tryptase IgE and IL-6 were not detectable. Exudation markers albumin, urea and uric acid were also detectable. The coefficient of variance (CV) values of the various cells and mediators varied between 13% and 137%. It was calculated that, except for the number of leukocytes and the concentration of ECP, it should be possible to detect ozone effects with a study-protocol of 6 repeated measurements among 35 children and an assumed 26% increase in cells or mediators per 100 micrograms O3 per m3. To measure increase in leukocytes number or in ECP concentration more children are needed. In conclusion, this pilot study has shown that it is possible to measure relevant biomarkers in NAL, and that these assays can be easily incorporated in epidemiological studies.

International Federation of Clinical Chemistry (IFCC) scientific division IFCC recommendation. Recommendation on sampling, transport and storage for the determination of the concentration of ionized calcium in whole blood, plasma and serum.

The substance concentration of ionized calcium (cCa2+) in blood, plasma or serum preanalytically may be affected by pH changes of the sample, calcium binding by heparin, and dilution by the anticoagulant solution. pH changes in whole blood can be minimized by anaerobic sampling to avoid loss of CO2, by measuring as soon as possible or by storing the sample in iced water to avoid lactic acid formation. cCa2+ and pH should be determined simultaneously. Plasma or serum: if centrifuged in a closed tube and measured immediately the pH of the sample will be close to the original value. If delay has occurred between centrifugation and the measurement, causing substantial loss of CO2, equilibration of the sample with a gas mixture corresponding to PCO2 = 5.3 kPa prior to the measurement is recommended. Conversion of the measured values to cCa2+ (7.4) is only valid if the pH is in the range 7.2-7.6 Ca2+ binding by heparin can be minimized by using either of the following: a final concentration of sodium or lithium heparinate of 15 IU/ml blood or less, by use of calcium titrated heparin with a final concentration less than 50 IU/ml blood. Dilution effect can be avoided by use of dry heparin in capillaries or syringes. When heparin solutions are used errors due to dilution or calcium binding can be reduced using syringes with a heparin solution containing free calcium ions corresponding to the mean concentration of ionized calcium in normal plasma. Conditions for blood collection, storage, and transport to avoid preanalytical errors are described.

Influence of ATP or oxygen plus substrate on occurrence of the calcium paradox.

Reperfusion of Ca2+-deprived rat hearts with Ca2+-containing medium results in irreversible cell damage (calcium paradox). In this study this type of cell damage was studied in the anoxic rat heart, in the presence and absence of glucose. Creatine kinase (CK) release was used to define cell damage. Hearts were perfused successively with Ca2+-containing medium (30 min), Ca2+-free medium (5 min), and Ca2+-containing medium (5 min). In the presence of glucose, myocardial ATP was maintained at a fairly high concentration. Reperfusion with Ca2+ resulted in an immediate and massive release of CK. In the absence of glucose, the ATP concentration was almost zero after 30 min. Reperfusion with Ca2+ did not result in release of CK. Massive release occurred as soon as these hearts were reoxygenated. It is concluded that this type of calcium-induced cell damage only occurs in the presence of ATP, or oxygen plus substrate. Mitochondria most likely play a major role in the occurrence of the calcium paradox because of their ability to accumulate huge amounts of Ca2+ under these conditions.

Are sodium bicarbonate and potassium bicarbonate fully dissociated under physiological conditions?

In solutions containing 160 mmol/l Na+ and K+, respectively, measurements with an ion-selective electrode system (KNA1, Radiometer), showed apparent falls in the respective Na+ and K+ concentrations when C1- was replaced by HCO3-. After correction for the change in liquid junction potential, the fall was 9.2 mmol/l for Na+ and 7.3 mmol/l for K+. On the basis of these findings we conclude that sodium bicarbonate and potassium bicarbonate are not fully dissociated in solution, and that NaHCO3(0) and KHCO3(0) do exist as chemical components with association constants of 0.72 and 0.55, respectively. Using these association constants, normal plasma will contain 1.2 mmol/l NaHCO3(0) and 0.03 mmol/l KHCO3(0). Thus NaHCO3(0) accounts for virtually the same amount of CO2 as the physically dissolved fraction. A review of all the currently known CO2 species in plasma suggests that there may be a residue of about 2 mmol/l of unknown CO2 species in normal plasma.

Rabbit model for estimating relative bioavailability, residues and tissue tolerance of intramuscular products: comparison of two ampicillin products.

A rabbit model for simultaneous investigation of the bioavailability, tissue residues and tissue tolerance of intramuscularly administered veterinary medicines is described. The bioavailability of ampicillin from two intramuscular products, which had been found to be different in calves, were compared in a two-way crossover design. The ampicillin levels in plasma, ampicillin residues in tissues, the plasma creatine kinase activity and the tissue damage at the injection sites were studied. The absolute bioavailabilities for the products were 100% and 40%. Differences in pharmacokinetics of ampicillin between sexes were observed after intravenous and intramuscular administration. Only slight tissue damage could be detected at the injection sites after intramuscular administration of these products. The results were compared with those obtained previously in calves and were found to be similar. Further investigations with other intramuscular drug products to validate this model are under way.

The calcium paradox: metabolic, electrophysiological, contractile and ultrastructural characteristics in four species.

The effect of transient depletion and repletion of calcium upon the electrophysiological activity, contractile activity, coronary flow, tissue energy metabolism and the cellular leakage of metabolites and creatine kinase has been characterized in the rat heart and now has been compared with the mouse, guinea pig and rabbit heart. The sudden and massive damage (the calcium paradox) which occurred in the rat was observed in all species studied. While fine differences between species were detectable, the basic principles and damaging effects of sudden transmembrane calcium fluxes would appear to be common to all species.

Some factors affecting a commercial kit for radioimmunoassay of digoxin using tritiated digoxin.

Some factors affecting results of digoxin determinations using one commercially available radioimmunoassay kit are described and discussed. Serum of pregnant women, cord blood, amniotic fluid and serum of patients taking spironolactone may show erroneously high digoxin activity due to lack of specificity of the antiserum. Cross-reaction with digitoxin was found to vary substantially with antibody-lot. Haemaccel (5 g/1) in the sample leads to too low results. When ethanol (100 g/1) is present results are too high. The need for testing the specifity of every new lot of antiserum before use is stressed.

Calcium paradox: changes in high-energy phosphate compounds of isolated perfused rat hearts.

When isolated rat hearts are perfused with Ca2+-containing medium, after a brief Ca2+-free period, irreversible cell damage occurs (calcium paradox). This phenomenon is concomitant with a rapid consumption of myocardial high-energy phosphate stores, prior to the appearance of these compounds in the effluent perfusion medium. A possible mechanism for the origin of myocardial necrosis, caused by intracellular Ca2+ overload, is discussed.

The effect of verapamil on the calcium paradox.

Reperfusion of isolated rat hearts with calcium-containing medium after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). Experiments were undertaken to determine whether the slow-channel calcium-antagonist drug verapamil protects calcium-deprived rat heart muscle against the consequences of readmitting calcium. Cell damage was quantitated in terms of creatine kinase (CK) release, depletion of endogenous creatine phosphate (CP) and adenosine triphosphate (ATP) stores, development of contracture as measured by longitudinal shortening of the left ventricle, and ultrastructural damage. Verapamil (1 mg/l) did not reduce the initial rate of CK release during reperfusion with calcium but reduced the initial rate at which myocardial CP and ATP stores were depleted and decreased the shortening of the longitudinal axis of the left ventricle. After 30 seconds of reperfusion the mean sarcomere length was significantly greater in the verapamil-treated hearts. These results can be interpreted to mean that inhibition of calcium inflex via the slow channels does not protect heart muscle against the deleterious effects of readmitting calcium after a period of calcium-free perfusion.

The effect of hypothermia on the occurrence of the calcium paradox.

Reperfusion of isolated rat hearts with Ca2+-containing medium, after a short Ca2+-free perfusion period, results in irreversible cell damage (calcium paradox). In this investigation the effect of hypothermia during (a) the Ca2+-free perfusion period and (b) the phase of reperfusion with Ca2+-containing medium was studied. Failure of the heart to recover mechanical activity, and creatine kinase release were used to define cell damage. Ca2+-free perfusion was performed at 37 degrees, 30 degrees, 25 degrees and 20 degrees C. Hypothermia during the Ca2+-free period was unable to prevent the calcium paradox. At 37 degrees C a Ca2+-free perfusion was perfusion period of 4 min was sufficient to lead to failure of the hearts to recover mechanical activity and to induce massive enzyme release upon reintroduction of Ca2+. At 20 degrees C the Ca2-free perfusion had to be continued for 25 min to induce failure to recover mechanical activity, and for 80 min to induce massive enzyme release upon reperfusion with Ca2+-containing medium. Hypothermia (10 degrees - 15 degrees C) during the reperfusion phase resulted in a moderate release of creatine kinase. Massive enzyme release occurred as soon as the temperature of the perfusate was raised above 25 degrees C.