Rat Whole Blood Research Articles

Oxygen transport properties in malaria-infected rodents--a comparison between infected and noninfected erythrocytes

This study was performed to investigate oxygen transport properties in whole blood (WB) of malaria-infected rats as well as in infected erythrocytes (IE) and noninfected erythrocytes (NIE) separated by density centrifugation. One week after inoculation with Plasmodium berghei, mean parasitemia was 26.5% and high correlations were found between parasitemia and hemoglobin concentration ([Hb]; r = -.902), mean cellular Hb concentration (MCHC; r = -.712), MetHb (r = .923), and base excess (r = -.922). Compared with control animals (C), the oxygen affinity was lower in WB under standard (pH 7.40) and simulated "in vivo" (pH 7.00) conditions (difference in P50, 5.7 and 5.1 mm Hg, respectively; 2P < .01, 2P < .05). In IE Hb and 2,3-biphosphoglycerate (2,3-BPG) concentrations were decreased (MCHC: IE 14.6 +/- 1.0, NIE 33.1 +/- 1.7 g/100 mL; [2,3-BPG]: IE 2.0 +/- 0.6, NIE 7.6 +/- 1.8 mmol/L), whereas [MetHb] and [ATP] were increased ([MetHb]: IE 19.0 +/- 3.7, NIE 0.7% +/- 0.8%; [ATP]: IE 33.5 +/- 2.4, NIE 6.2 +/- 1.0 mumol/g Hb). At pH 7.40, half-saturation oxygen tension (P50) was reduced in IE (29.6 +/- 2.6, NIE 39.2 +/- 5.4 mm Hg, 2P < .001), which correlates with lower [2,3-BPG], increased MetHb content, and higher intrinsic Hb-O2 affinity. However, at pH 7.00, the oxygen affinity was lower in IE when compared with NIE, which was most likely due to high [ATP] in IE. The resulting Bohr coefficients (BC) calculated for CO2 and lactic acid were extremely high in IE and low in NIE (at 50% O2-saturation BCCO2: IE -1.04 +/- 0.06, NIE -0.26 +/- 0.10, 2P < .001; BCLac: IE -0.82 +/- 0.16, NIE -0.47 +/- 0.07, 2P < .001), which was caused by different [2,3-BPG] and [ATP] as well as probably by structural changes of the Hb molecule. The O2 capacity was 14.1 mL per 100 mL erythrocytes in IE compared with 44.4 mL/100 mL in NIE. On the basis of the calculated arterio-venous O2 difference under "in vivo" conditions, the infected red blood cell fraction transports 30% of the O2 amount delivered to the tissues by the noninfected cells (IE 8.0, NIE 26.9 mL/100 mL red blood cells). We conclude that the O2 transport in malaria infected blood is not only affected by the degree of anemia but also by the percentage of infected erythrocytes.

Stress and/or tranylcypromine treatment affects serotonergic measures in blood and brain in rats

Since stress can alter serotonin (5-hydroxytryptamine, 5-HT) turnover in the brain and the periphery, the effects of different types of acute stress on serotonin and related substances in the whole blood and various brain areas in rats pretreated with tranylcypromine (TCP) were studied. TCP administered alone caused a rise in 5-HT, a fall in its metabolite (5-hydroxyindoleacetic acid, 5-HIAA) in the whole blood and in every part of the brain analyzed relative to controls. In rats given TCP and subjected to footshock or water-immersion restraint stress similar changes, but to a different extent, were observed. 5-HT level remained essentially constant except in the blood and the limbic system, whereas 5-HIAA level was found to be increased in the blood and the brain, mainly in the limbic system and the brainstem following footshock. Water-immersion restraint stress caused an increase in 5-HT only in the limbic system without any changes in 5-HT and 5-HIAA in the blood. Relative to controls, an increase in total tryptophan concentration in the whole blood and in every part of the brain was found only after footshock application with or without pretreatment with TCP. In conclusion, responses to stress in rats may depend upon the type of stimulus applied as well as of a concurrent administration of TCP. Some regional differences may account for an altered in vivo efficacy of this drug.

High-performance liquid chromatographic determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) and its deacetyl metabolite in plasma, whole blood, urine and tissue samples in rats

A reversed-phase high-performance liquid chromatographic method was developed for the determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648, I), a new rifamycin derivative, and its 25-deacetyl metabolite (KRM-1671, II) in plasma, whole blood, tissues and urine from rats. I and II were coextracted with an internal standard from each sample matrix by solid-phase extraction (Bond Elut). Plasma and urine were directly loaded onto Bond Elut, while whole blood and tissues were homogenized and extracted with methanol or dichloromethane—chloroform prior to Bond Elut extraction. The extracts were chromatographed on Shim-pack CLC-ODS(M) using acetonitrile—0.02 M citrate buffer containing 0.1 M sodium perchlorate (2:1, v/v), and peaks were detected at 643 nm. The validation data showed that the assays for I and II in plasma, whole blood, tissues and urine were selective, accurate and reproducible.

Isologous and heterologous platelet aggregation induced by tumor cells from three murine mammary gland adenocarcinomas with different metastatic abilities.

The addition of neoplastic cells (NC) isolated from murine mammary gland adenocarcinomas having moderate and high metastatic ability on in vitro heparinized mice platelet rich plasma (PRP) induced platelet aggregation. One non-metastatic line did not induce aggregation. The aggregatory effect was not correlated with the metastatic abilities. None of the three types of tumors did cause aggregation on whole blood or PRP of rats. These adenocarcinomas seem to be suitable models to study in vitro the interactions between platelets and tumor cells having metastatic and non-metastatic potentials.

Serotonergic systems in brain and blood under stress and tranylcypromine treatment in rats

The effects of stress on serotonin (5-hydroxytryptamine, 5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), and/or tryptophan in whole blood and various brain areas of rats pretreated with tranylcypromine were studied. In the whole blood, tranylcypromine given alone caused a rise in levels of 5-HT and a fall in levels of its metabolite (5-HIAA) and the ratio of 5-HIAA:5-HT, whereas in stressed rats pretreated with tranylcypromine only the last two findings were observed. We found that animals given tranylcypromine and subjected to water-immersion restraint stress exhibited the greatest rise in 5-HT levels in midbrain, hippocampus, and hypothalamus, together with the lowest 5-HIAA:5-HT ratio relative to controls, whereas in cortex, cerebellum, medulla, and striatum the highest levels of 5-HT together with the lowest ratio of 5-HIAA:5-HT were observed after the administration of tranylcypromine alone. 5-HT levels were found to be higher in medulla and striatum in rats given tranylcypromine alone relative to stressed rats pretreated with this drug. We concluded that regional differences account for variable effects of tranylcypromine on blood and brain Serotonergic systems in vivo.

Measurement of adducts of benzoquinone with hemoglobin and albumin.

Benzoquinones (BQ) are genotoxic species that stem from metabolism of phenolic compounds. We have developed a method for measuring adducts of 1,2- and 1,4-benzoquinone (1,2-BQ and 1,4-BQ) with cysteine residues of hemoglobin (Hb) and albumin (Alb). The method employs a reductive catalyst (Raney nickel) to selectively cleave sulfur-bound adducts so that they may be extracted with an organic solvent, derivatized with heptafluorobutyrylimidazole and measured by GC-ECD or GC-MS. Reactions of 1,4-BQ (0-300 microM) with whole blood of both F344 rats and humans resulted in linear formation of adducts with Hb and Alb. Adducts of 1,2-BQ with human Alb were formed by activation of catechol (0-300 microM) in situ with horseradish peroxidase. The mean half lives in vitro of 1,4-BQ in blood from humans and rats were 3.5 and 0.68 h, respectively. Second-order rate constants for reaction of 1,4-BQ with Hb and Alb in whole blood were estimated to be 18 and 76 l/mol/h for humans and 180 and 74 l/mol/h for rats respectively. Interestingly, the following high background levels of 1,2-BQ and 1,4-BQ adducts were observed in the proteins: 1,2-BQ-human Hb = 1.5 nmol/g, rat Hb = 25.1 nmol/g; human Alb = 8.0 nmol/g; 1,4-BQ-human Hb = 27.2 nmol/g, rat Hb = 11.5 nmol/g; human Alb = 20.5 nmol/g. These large background levels of BQ adducts suggest that significant exposure to BQ precursors occurs from dietary and/or endogenous sources. Given current evidence regarding the genotoxicity of BQ, such exposures and their health consequences should be investigated.

Changes in Platelet Aggregation in Whole Blood, Plasma and Washed Platelets in Streptozotocin-induced Diabetic Rats: Time-dependent Change in the Antiaggregatory Activity of Diabetic Rat Plasma

Time-dependent changes in platelet aggregation in whole blood, platelet-rich plasma (PRP) and washed platelets were studied in streptozotocin-induced diabetic rats. Collagen-induced aggregation of whole blood and PRP from diabetic rats were significantly reduced within 8 weeks after induction of diabetes, although that in washed platelets were increased from 8 weeks. Plasma from diabetic rats within 8 weeks attenuated platelet aggregation, whereas diabetic plasma at 12 weeks showed no inhibitory effect. Insulin treatment normalized aggregation in whole blood and PRP and abolished the antiaggregatory activity of diabetic plasma. These results suggest the plasma antiaggregating activity appears in the early stage of diabetes, which may contribute to the hypoaggregation in whole blood and PRP. The inhibitory activity disappeared in the later stage. Plasma factor(s) accounting for the antiaggregatory effect of diabetic plasma has not yet characterized.

Studies on the Biological Effects of Rare Earth Elements. IV. Effects of Chlorides of Dy, Eu, Yb and Y on Rare Earth Elements, Ca, Mg and P Contents in Various Organs in the Rat after Intravenous Administration.

Effects of rare earth elements (REEs) on the time courses of REE, Ca, Mg and P concentrations in the liver, kidney, spleen, lung, femur and whole blood were investigated in the male rats of Wistar strain. Four kinds of REE salts, i.e. dysprosium chloride (DyCl3), europium chloride (EuCl3), ytterbium chloride (YbCl3) and yttrium chloride (YCl3)], were administered intravenously from the caudal vein (10 mg/kg as REE). Contents of REE, Ca, Mg and P were estimated by the wet digestion-ICP-AES method as a function of time. Results were as follows : 1) Concentrations of Ca in the liver, spleen and lung increased extremely in the rat administered with YCl3. Pattern of hepatic Ca concentration as a function of time was similar to that of hepatic Y concentration, and Ca concentration was not significantly different from the control group at 45th d after the administration. Patterns of Ca concentrations were different from Y in the spleen and lung. REEs administration did not affect significantly the concentrations of Ca, Mg and P in the liver, kidney, spleen and lung except the concentrations of Ca in the liver, spleen and lung of Y-administered rats. 2) Concentrations of Ca, Mg and P in the whole blood of REE-administered rats varied from the control group within 1 d after the administration. 3) No significant effects were observed in the femoral Ca, Mg and P concentrations. 4) Concentrations of REEs in the whole blood and liver decreased exponentially in the ranges of 15 min-1 d and 1-45 d (8 h-45 d in case of YCl3-administered rats) respectively. The half lives of Dy, Eu, Yb and Y were calculated as 6.94, 6.78, 6.84 and 0.427 h for the whole blood, and 16.2, 21.0, 15.5 and 19.3 d for the liver, respectively.

Mechanism of ethanol-induced aggregation in whole blood

Effects of ethanol on blood clotting and platelet aggregation have been reported in many models, but its in vitro actions in whole blood, impedance aggregometry have not been reported. We investigated the effect of ethanol in vitro in whole blood and platelet rich plasma of humans and rats, as measured by impedance aggregometry. Ethanol (34 to 170 mM) induced concentration-dependent aggregation in whole blood but not platelet rich plasma. In further studies in rats, aggregation was inhibited by pretreatment of whole blood with the prostacyclin analog iloprost or the enzyme apyrase, which degrades ADP to AMP. Levels of ethanol which produced aggregation in whole blood were also associated with concentration-dependent hemolysis. Based on the requirement for whole blood for ethanol-induced aggregation, the inhibitory effect of apyrase and our observation of hemolysis, and previous studies which have demonstrated the potential contribution of ADP from lysed red blood cells to platelet aggregation, we conclude that ethanol-induced aggregation in whole blood is mediated by erythrocyte lysis and the ADP released from these cells.

The Influence of Hemodilution in Normal and Cirrhotic Rats in Relation to Hepatic Energy Metabolism

The effects of acute hemodilution on hepatic energy status were compared between carbon-tetrachloride-induced cirrhotic rats and normal rats, using arterial blood ketone body ratio and hepatic energy charge as indices. Arterial blood was withdrawn and replaced with rat plasma in hemodiluted rats and with whole blood in sham-diluted control rats. In normal rats, arterial blood ketone body ratio and energy charge level at 6 hours after hemodilution decreased when the hematocrit value was less than 15%. In contrast, the values decreased at an even higher hematocrit level in cirrhotic rats. It was suggested from these results that the hemodilution procedure exerts a more profound hypoxic effect at a milder hemodilution on a cirrhotic liver than on a normal liver.

High-performance liquid chromatographic analysis, plasma protein binding and red blood cell partitioning of phenprobamate.

A new high-performance liquid chromatographic procedure for the analysis of phenprobamate, a skeletal muscle relaxant in biologic fluids was developed. The method used a C18 reverse phase column, a mobile phase of methanol/acetonitrile/water (33:15:52), and UV detection at 215 nm. The assay procedure was applied to the determination of phenprobamate binding to rat and human plasma proteins using the equilibrium dialysis method. In addition, the red blood cell/plasma partitioning was determined in the whole blood of rats and humans. Phenprobamate exhibited a moderate binding to plasma proteins of rat (74.3 +/- 2.2 per cent) and human (80.5 +/- 1.1 per cent). The protein binding was concentration-independent in the range of 10 to 80 micrograms ml-1. Phenprobamate binding to plasma proteins was also determined in the presence of 10 micrograms ml-1 acetaminophen. The protein binding of phenprobamate was not significantly altered by acetaminophen (74.4 +/- 0.6 per cent for rat plasma; 75.7 +/- 1.6 per cent for human plasma). The distribution ratios of phenprobamate between the red blood cells and plasma were greater than unity, 1.86 and 1.59 in rat and human, respectively, indicating a preferential partitioning of the drug in the red blood cells.

The Influence of Hemodilution in Normal and Hepatectomized Rats in Relation to Hepatic Energy Metabolism

The effects and safety limits of acute hemodilution on hepatic energy status were determined by comparison of arterial blood ketone body ratio (acetoacetate/3-hydroxybutyrate) and hepatic energy charge levels in 70% hepatectomized rats and nonhepatectomized rats. Arterial blood was withdrawn and replaced with rat plasma in hemodiluted rats and with whole blood in sham-diluted control rats. The arterial blood ketone body ratio at 6 hours after hemodilution in hepatectomized and sham-diluted rats (0.454±0.037) was lower than that of nonhepatectomized and sham-diluted rats (0.686±0.044). In hepatectomized groups, it decreased to 0.403±0.043 when the hematocrit value was less than 20% and to 0.286±0.031 when it was less than 15%. In hepatectomized rats, hepatic energy charge at 6 hours after hemodilution was 0.809±0.021 in sham-diluted controls, but decreased to less than 0.8 when the hematocrit was less than 20%. On the other hand, in nonhepatectomized rats, the energy charge was greater than 0.8 even when the hematocrit was less than 15%. These results suggest that hemodilution procedure exerts more profound hypoxic effect on the remnant liver than on the normal one.

Analysis of whole blood manganese by flameless atomic absorption spectrophotometry and its use as an indicator of manganese status in animals

To investigate the use of whole blood manganese (Mn) as an indicator of total body Mn, we measured Mn in whole blood and liver of rats fed purified diets containing adequate (45 μg Mn/g diet) or deficient (1 μg Mn/g diet) Mn. The mean hepatic Mn concentration was significantly lower ( P < 0.001) in the Mn-deficient group compared to the control group, 0.36, μg Mn/g and 1.73 μg Mn/g, respectively. Furthermore, whole blood Mn was significantly reduced ( P < 0.001) in the deficient group when compared to the control group, 4.0 ng Mn/ml and 8.6 ng Mn/ml, respectively. Hepatic Mn linearly regressed against whole blood Mn yielded a statistically significant ( P < 0.001) correlation coefficient of 0.775. These data suggest that whole blood Mn is a valid indicator of body Mn status and thus may be useful, in addition to the measurements of serum copper and zinc, for the diagnosis and prognosis of diseases in which the metabolism of trace elements is affected. In addition, this paper describes and delineates operational parameters for the measurement of whole blood Mn using the IL 551 atomic absorption spectrophotometer and the IL 555 B flameless atomizer.

Effect of diclofenac sodium on the arachidonic acid cascade

The anti-inflammatory activity of nonsteroidal anti-inflammatory drugs Is primarily attributed to inhibition of distinct steps in the arachidonic acid cascade, particularly, the cyclo-oxygenase pathway. Diclofenac sodium, a compound of this class of drugs, appears to have a dual effect since it also regulates the lipoxygenase pathway. Study of appropriate cell systems (leukocytes and whole blood in rats) demonstrates that diclofenac's potent inhibition of cyclooxygenase activity causes a sharp reduction in the formation of prostaglandin, prostacyclin, and thromboxane products, all key mediators of inflammation. Recent work discloses that at higher concentrations, diclofenac sodium also reduces the formation of products of the lipoxygenase pathway (5-hydroxyeicosatetraenoic acid, leukotrienes). The mechanism by which this evolves, however, appears to be unrelated to direct inhibition of lipoxygenase. Instead, by enhancing its reincorporation into triglycerides, diclofenac sodium reduces the intracellular level of free arachidonic acid.

Haematological, immunological and endocrinological aspects of chronic high frequency blood sampling in rats with replacement by fresh or preserved donor blood.

Haematological, immunological and endocrinological aspects of blood transfusions with either freshly collected or preserved donor blood were investigated in chronically cannulated unrestrained rats. Three anticoagulant preservatives were tested: citrate, citrate-dextrose and citrate-phosphate-dextrose-adenine (CPDA-1). Prolactin was used as an indicator of stress in endocrine studies. The repeated collection of 4 ml blood at 2-week intervals did not affect normal blood composition. Whole blood of rats could be stored in citrate, citrate-dextrose or CPDA-1 for 8, 22 or 35 days, respectively. Blood transfusions with fresh or preserved donor blood of F1 (R X U) rats did not affect normal blood composition nor did it induce immunological responses in F1 rats. Frequent blood sampling for several hours at highest rates of 1 sample/min did not affect prolactin secretion when blood volume reduction was replaced by blood transfusions with fresh donor blood. However, compensation with preserved blood affected prolactin secretion significantly. Blood transfusions did not affect health, behaviour, cyclicity or pseudopregnancy. The application of blood transfusion in chronically cannulated rats appeared to be quite simple. Its advantages are the possibility of following individual secretion patterns of blood-bound substances, the repeated use of animals and the reduction of the number of animals.

Monitoring of aluminum in whole blood, plasma, serum, and water by a single procedure using flameless atomic absorption spectrophotometry.

A simple, time-saving procedure to measure aluminum (AI) in whole blood, plasma, and water samples of low ionic strength by flameless atomic absorption spectrometry is presented. With this procedure, an analagous pretreatment is given to all samples. Moreover, the pretreated samples are analyzed using an identical program of the atomic absorption spectrophotometer. The detection limit is 1.3 micrograms/L for aqueous solutions and 1.9 micrograms/L, 1.8 micrograms/L, and 2.3 microL for serum, plasma, and blood samples respectively. The precision of the method varies between 2.8% and 4.2% of all analyses. Using this method, a comparison was made between Al levels in whole blood (AlB) and plasma (AlP) in rats and AlB and AlP in renal patients. The AlB and AlP were strongly correlated. It is concluded that the monitoring of AlB may have a similar prognostic value for toxicity as the monitoring of AlP, but more experimental advantages.

Comparison of taurine-verapamil interaction in hamsters and rats

1. Taurine concentrations were determined in hearts and whole blood of female Syrian golden hamsters and Wistar rats following 30 days of drinking: (i) tap water, (ii) taurine solution or (iii) a taurine uptake inhibitor (GES). 2. The groups were duplicated in animals receiving daily verapamil injections during the treatment period. 3. Heart taurine increased with (ii) and decreased with (iii) in both species; blood taurine changed similarly in rats only. 4. Verapamil enhanced taurine uptake and offset the GES inhibition in the hamster heart, but had no such effect in rats. 5. Possible implications of the species differences are elaborated upon.

Quantification of imipramine and its major metabolites in whole blood, brain, and other tissues of the rat by liquid chromatography.

An assay procedure is presented using high-performance liquid chromatography and fluorescence detection for simultaneous determination of imipramine, desipramine, and their 2-hydroxylated metabolites in whole blood and various tissues of the rat. By modifying methods previously described for plasma, we have developed an assay method for sensitive, reproducible quantitation of these compounds in rat plasma, whole blood, brain, liver, and fetus. Increasing the volume of the extraction solvent (20% butanol in hexane) increased recovery of imipramine and desipramine to greater than 90% for all tissues examined. No interfering peaks were observed for any of the tissues studied. Addition of n-butylamine as a modifier to the mobile phase decreased the retention of all four compounds and decreased peak tailing of the demethylated compounds. The total elution time was less than 15 min. Using these methods the coefficient of variation for all four compounds was generally less than 5% for each of the five tissues examined. The calibration curves were linear for standard concentrations of 25-2,000 ng/ml spiked tissue homogenate. This method has sufficient sensitivity and specificity for use in pharmacokinetic studies of imipramine.

Lack of differences in blood and tissue concentrations of endogenous ethanol in conventional and germfree rats

Headspace gas chromatography was used to determine the concentrations of endogenous ethanol in blood and tissue of conventional and germfree rats. In all biological specimens analysed, the four principal volatile endogenous substances were identified as methanol, acetaldehyde, ethanol and acetone. No statistically significant differences in the concentrations of endogenous ethanol were noted between conventional and germfree animals. In whole blood, liver, kidney, and brain of germfree rats the concentrations of endogenous ethanol were 4.2±0.19 μM, 5.1±0.55 μM, 8.2±0.59 μM and 4.4±0.17 μM (means±SE), respectively. The higher concentration in kidney was also observed in conventional rats. Our results suggest that ethanol is a normal metabolic intermediate in rats and does not exclusively arise from microbial fermentation reactions in the gastrointestinal tract.

Determination of Benzene, Phenol, Catechol, and Hydroquinone in Whole Blood of Rats and Mice

Determination of Benzene, Phenol, Catechol, and Hydroquinone in Whole Blood of Rats and Mice