Investigation of dose-dependent effects of berberine against renal ischemia/reperfusion injury in experimental diabetic rats / Investigación de los efectos dependientes de dosis de la berberina contra la lesión de isquemia/reperfusión renal en ratas diabéticas experimentales

BACKGROUND: Ischemia-reperfusion injury causes various severe morphological and functional changes in diabetic patients. To date, numerous antidiabetic and antioxidant agents have been used for treatment of the disease-related changes. OBJECTIVES: We aimed to examine effective therapeutic doses or doses of berberine against renal ischemia/reperfusion injury (IRI) in a streptozotocin (STZ)-induced diabetic rat model by histopathological and biochemical analysis. METHODS: Thirty male Sprague Dawley rats were treated with STZ injection for the development of diabetes, and divided into the following groups: STZ-induced diabetic group (STZ); IRI-induced diabetic group (STZ + IRI); 50 mg/kg berberine (BRB) treated diabetic group after inducing IRI (STZ + IRI + BRB1); 100 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB2); 150 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB3). Bilateral renal ischemia model was applied for 45min, then reperfusion was allowed for 14 days in STZ-induced diabetic rats. Renal injury was detected histopathologically. Blood urea nitrogen (BUN), creatinine and lactate dehydrogenase (LDH) levels were measured in serum using the ELISA method. Total antioxidant status (TAS) and total oxidant status (TOS) of renal tissue was studied by spectrophotometric assay. Oxidative stress index (OSI) was calculated as TOS-to-TAS ratio. Tumor necrosis factor alpha (TNF-alfa), C-reactive protein (CRP), Na+/K+-ATPase (sodium pump), and Ca2+-ATPase (calcium ATPase) enzyme levels were measured in tissues using the ELISA method. Anti-apoptotic Bax and pro-apoptotic Bcl-2 protein levels were detected by Western blot analysis. All data were evaluated statistically. RESULTS: The highest histopathological score was detected in the STZ+IRI group compared to the other group. BRB administration at the doses of 100mg/kg and 150 mg/kg markedly improved renal injury. BUN and creatinine levels significantly increased in the STZ+IRI group compared to the STZ group (p < 0.001). 100 mg/kg and 150 mg/kg BRB administration significantly decreased those levels (p < 0.01). The highest TOS and the lowest TAS levels were detected in the STZ+IRI group (p < 0.001). IRI markedly aggravated inflammation via increasing levels of TNF-Alpha and CRP (< 0.001), and caused apoptosis via inducing Bcl-2 protein, and suppressing Bax protein (p < 0.001). BRB administration at the doses of 100 mg/kg and 150 mg/kg showed anti-oxidant, anti-inflammatory and anti-apoptotic effects (p < 0.01). The LDH enzyme, was used as a necrosis marker, was higher in the STZ+IRI group than other groups. BRB administration at all of the doses, resulted in the decline of LDH enzyme level (p < 0.001). Ca2+-ATPase and Na+/K+-ATPase enzyme activities decreased in the STZ+IRI group compared to the STZ group (p < 0.001), while BRB administration at the doses of 100 mg/kg and 150mg/kg significantly increased those of enzyme activities, respectively (p < 0.05). CONCLUSION: Ischemia with diabetes caused severe histopathological and biochemical damage in renal tissue. The high doses of berberine markedly improved histopathological findings, regulated kidney function via decreasing BUN and creatinine levels, and rearranged intercellular ion concentration via increasing Na+/K+-ATPase and Ca2+- ATPase levels. Berberine showed anti-oxidant, anti-apoptotic, and anti-inflammatory effects. According to these data, we suggest that berberine at the doses of 100 and 150 mg may be used as a potential therapeutic agent to prevent renal ischemic injury

ANTECEDENTES: La reperfusión de la isquemia provoca graves cambios morfológicos y funcionales en los pacientes diabéticos. Hasta la fecha numerosos antidiabéticos y agentes antioxidantes han sido utilizados para el tratamiento de los cambios relacionados con la enfermedad. OBJETIVOS: El objetivo fue examinar las dosis terapéuticas efectivas o las dosis de berberina (BRB) frente a la insuficiencia renal por isquemia/reperfusión (IRI) en estreptozotocina (STZ) inducida por el modelo de rata por análisis histopatológico y bioquímico. MÉTODOS: Treinta ratas machos Spraque-Dawley fueron tratadas con inyección de STZ para el desarrollo de diabetes, se dividieron en los siguientes grupos: grupo diabético inducido por STZ; grupo diabético inducido por IRI (STZ + IRI); grupo diabético tratado con 50 mg/kg de BRB después de la inducción de IRI (STZ + IRI + BRB1); grupo diabético tratado con 100 mg/kg de BRB después de IRI (STZ + IRI + BRB2), y grupo diabético tratado con 150 mg/kg de BRB después de IRI (STZ + IRI + BRB3). Se aplicó un modelo de isquemia renal bilateral durante 45min, luego se permitió la reperfusión durante 14 días en ratas diabéticas inducidas por STZ. La lesión renal fue detectada histopatológicamente. Los niveles de nitrógeno ureico en sangre (BUN), creatinina y lactato deshidrogenasa (LDH) se midieron en suero por el método ELISA. El estado antioxidante total (TAS) y el estado oxidante total (TOS) del tejido renal se estudiaron mediante un ensayo espectrofotométrico. El índice de estrés oxidativo (OSI) fue calculado como la relación TOS-a-TAS. El factor de necrosis tumoral alfa (TNF-alfa), proteína C reactiva (CRP), Na+/K+-ATPasa (bomba de sodio) y la Ca2+-ATPasa (calcio ATPasa) de la enzima se midieron los niveles en los tejidos mediante el método ELISA. Los niveles de proteína anti-apoptótica Bax y pro-apoptótica Bcl-2 se detectaron por el análisis de Western blot. Todos los datos fueron evaluados estadísticamente. RESULTADOS: La mayor puntuación histopatológica fue detectada en el grupo STZ + IRI en comparación con el otro grupo. La administración de BRB en dosis de 100 y 150 mg/kg mejoró notablemente la lesión renal. Los niveles de BUN y creatinina aumentaron significativamente en el grupo STZ+IRI en comparación con el grupo STZ (p < 0,001). La administración de 100 y 150 mg/kg de BRB disminuyó significativamente esos niveles (p < 0,01). Los TOS más altos y los niveles más bajos de TAS se detectaron en el grupo STZ+IRI (p < 0,001). El IRI agravó notablemente la inflamación a través del aumento de los niveles de TNF-alfa y de PCR (< 0,001), y causó la apoptosis a través de la inducción de la proteína Bcl-2 y la supresión de la proteína Bax (p < 0,001). La administración de BRB con las dosis de 100 y 150mg/kg mostró efectos antioxidante, antiinflamatorio y antiapoptóticos (p < 0,01). La enzima LDH que se usó como marcador de necrosis fue mayor en el grupo STZ + IRI que en los otros grupos. La administración de BRB en todas las dosis, dio lugar a una disminución del nivel de la enzima LDH (p < 0,001). Las actividades de las enzimas Ca2+-ATPasa y Na+/K+-ATPasa disminuyeron en el grupo STZ + IRI en comparación con el grupo STZ (p < 0,001, mientras que la administración de BRB en dosis de 100 y 150 mg/kg incrementó significativamente las actividades de las enzimas, respectivamente (p < 0,05). CONCLUSIÓN: La isquemia con diabetes causó graves daños histopatológicos y bioquímicos en el tejido renal. Las altas dosis de BRB mejoraron notablemente los hallazgos histopatológicos, la función renal regulada a través de la disminución de los niveles de BUN y creatinina, y la concentración de iones intercelulares reordenados mediante el aumento de los niveles de Na+/K+-ATPasa y la Ca2+-ATPasa. La BRB mostró efectos antioxidantes, antiapoptóticos y antiinflamatorios. De acuerdo con estos datos, sugerimos que la BRB en dosis de 100 y 150 mg se puede usar como un agente terapéutico potencial para prevenir la lesión isquémica renal

Investigation of dose-dependent effects of berberine against renal ischemia/reperfusion injury in experimental diabetic rats.

BACKGROUND: Ischemia-reperfusion injury causes various severe morphological and functional changes in diabetic patients. To date, numerous antidiabetic and antioxidant agents have been used for treatment of the disease-related changes. OBJECTIVES: We aimed to examine effective therapeutic doses or doses of berberine against renal ischemia/reperfusion injury (IRI) in a streptozotocin (STZ)-induced diabetic rat model by histopathological and biochemical analysis. METHODS: Thirty male Sprague Dawley rats were treated with STZ injection for the development of diabetes, and divided into the following groups: STZ-induced diabetic group (STZ); IRI-induced diabetic group (STZ+IRI); 50mg/kg berberine (BRB) treated diabetic group after inducing IRI (STZ+IRI+BRB1); 100mg/kg BRB treated diabetic group after IRI (STZ+IRI+BRB2); 150mg/kg BRB treated diabetic group after IRI (STZ+IRI+BRB3). Bilateral renal ischemia model was applied for 45min, then reperfusion was allowed for 14 days in STZ-induced diabetic rats. Renal injury was detected histopathologically. Blood urea nitrogen (BUN), creatinine and lactate dehydrogenase (LDH) levels were measured in serum using the ELISA method. Total antioxidant status (TAS) and total oxidant status (TOS) of renal tissue was studied by spectrophotometric assay. Oxidative stress index (OSI) was calculated as TOS-to-TAS ratio. Tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), Na+/K+-ATPase (sodium pump), and Ca2+-ATPase (calcium ATPase) enzyme levels were measured in tissues using the ELISA method. Anti-apoptotic Bax and pro-apoptotic Bcl-2 protein levels were detected by Western blot analysis. All data were evaluated statistically. RESULTS: The highest histopathological score was detected in the STZ+IRI group compared to the other group. BRB administration at the doses of 100mg/kg and 150mg/kg markedly improved renal injury. BUN and creatinine levels significantly increased in the STZ+IRI group compared to the STZ group (p<0.001). 100mg/kg and 150mg/kg BRB administration significantly decreased those levels (p<0.01). The highest TOS and the lowest TAS levels were detected in the STZ+IRI group (p<0.001). IRI markedly aggravated inflammation via increasing levels of TNF-α and CRP (<0.001), and caused apoptosis via inducing Bcl-2 protein, and suppressing Bax protein (p<0.001). BRB administration at the doses of 100mg/kg and 150mg/kg showed anti-oxidant, anti-inflammatory and anti-apoptotic effects (p<0.01). The LDH enzyme, was used as a necrosis marker, was higher in the STZ+IRI group than other groups. BRB administration at all of the doses, resulted in the decline of LDH enzyme level (p<0.001). Ca2+-ATPase and Na+/K+-ATPase enzyme activities decreased in the STZ+IRI group compared to the STZ group (p<0.001), while BRB administration at the doses of 100mg/kg and 150mg/kg significantly increased those of enzyme activities, respectively (p<0.05). CONCLUSION: Ischemia with diabetes caused severe histopathological and biochemical damage in renal tissue. The high doses of berberine markedly improved histopathological findings, regulated kidney function via decreasing BUN and creatinine levels, and rearranged intercellular ion concentration via increasing Na+/K+-ATPase and Ca2+- ATPase levels. Berberine showed anti-oxidant, anti-apoptotic, and anti-inflammatory effects. According to these data, we suggest that berberine at the doses of 100 and 150mg may be used as a potential therapeutic agent to prevent renal ischemic injury.

Effect of magnesium supplementation on blood rheology in NOS inhibition-induced hypertension model.

This study investigated the effects of magnesium on blood rheological properties and blood pressure in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension was induced by oral administration of the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg/day) for 6 weeks and systolic blood pressure was measured by the tail-cuff method. The groups receiving magnesium supplementation were fed with rat chow containing 0.8% magnesium oxide during the experiment. At the end of experiment, blood samples were obtained from abdominal aorta, using ether anesthesia. Plasma and erythrocyte magnesium levels were determined by the atomic absorption spectrometer. RBC deformability and aggregation were determined by rotational ektacytometry. Plasma fibrinogen concentration was evaluated by ELISA. Whole blood and plasma viscosities were determined by viscometer and intracellular free Ca++ level was measured by using spectroflurometric method. Blood pressure was elevated in hypertensive groups and suppressed by magnesium therapy. Plasma viscosity and RBC aggregation were found to be higher in hypertensive rats than control animals and these parameters significantly decreased in magnesium supplemented hypertensive animals. Other measurements were not different between experimental groups. These results confirm that blood pressure, plasma viscosity and RBC aggregation increased in NOS inhibition-induced hypertension model and oral magnesium supplementation improved these parameters.

Peripheral mononuclear leukocyte DNA damage, plasma prolidase activity, and oxidative status in patients with benign prostatic hyperplasia.

OBJECTIVES: Prolidase plays a major role in collagen turnover, matrix remodeling, and cell growth. Benign prostatic hyperplasia (BPH) may be associated with an increased extracellular matrix deposition. Therefore, the present study was designed to investigate the plasma prolidase activity, oxidative status, and peripheral mononuclear leukocyte DNA damage in patients with BPH. PATIENTS AND METHODS: Twenty-six male patients with BPH and 24 healthy male subjects were included in this study. Blood samples were collected from antecubital vein after an overnight fasting period, and the plasma was separated. Plasma prolidase activity, total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) were determined. The peripheral lymphocyte oxidative DNA damage was determined using an alkaline single cell gel electrophoresis assay (comet assay). RESULTS: The plasma prolidase activity, TOS levels, OSI values, and peripheral mononuclear leukocyte DNA damage were significantly higher (P < 0.001), while the TAC levels were significantly lower (P < 0.001) in patients with BPH than controls. In BPH patients, the prolidase activity was significantly associated with TAC levels (r = -0.366, P < 0.05), TOS levels (r = 0.573, P < 0.001), and OSI (r = 0.618, P < 0.001) and peripheral mononuclear leukocyte DNA damage (r = 0.461, P < 0.001). CONCLUSIONS: Our results showed that BPH might be associated with an increased oxidative stress, and also an increased plasma prolidase activity. Increased prolidase activity might play an important role in the etiopathogenesis and/or progression of BPH.

Effect of lanthanides on red blood cell deformability and response to mechanical stress: role of lanthanide ionic radius.

Prior studies exploring the effects of lanthanides (Ln) on red blood cells (RBC) have primarily focused on ion transport, cell fusion, and membrane protein structure. Our previous report [Biorheology 44 (2007), 361-373] dealt only with lanthanum (La) and cell rigidity; the present study extends these observations to other lanthanides (Nd, Sm, Eu, Dy, Er) and to RBC response to mechanical shear. Deformation-shear stress behavior of normal human RBC was measured at Ln concentrations up to 200 µM. In another series of experiments, RBC were exposed to mechanical stress (190 Pa, 300 s) at 50 µM Ln and deformation-stress data obtained prior to and after this stress. Data were fitted to a Lineweaver-Burke model to obtain the shear stress at one-half maximum deformation (SS1/2). Our results include: (1) lanthanides cause decreased cell deformability with the magnitude of the decrease dependent on concentration and shear stress; (2) this decrease of deformability is affected by Ln ionic radius such that La>Nd>Sm>Eu>Dy>Er and is reversible for cells in Ln-free media; (3) mechanical stress decreases deformability (i.e., increases SS1/2) such that compared to control, La and Sm reduce and Dy and Er enhance the mechanical stress effect; (4) the decrease of deformability consequent to mechanical stress scales inversely with Ln ionic radius. These results indicate a reciprocal relation between cell rigidity and sensitivity to mechanical stress that is mediated by Ln ionic radius. Additional studies are clearly warranted, particularly those that explore membrane-glycocalyx and intracellular mechanisms.

Wavelength selection in measuring red blood cell aggregation based on light transmittance.

The reversible aggregation of red blood cells (RBC) is of current basic science and clinical interest. Using a flow channel and light transmittance (LT) through RBC suspensions, we have examined the effects of wavelength (500 to 900 nm) on the static and dynamic aspects of RBC aggregation for normal blood and suspensions with reduced or enhanced aggregation; the effects of oxygenation were also explored. Salient observations include: 1. significant effects of wavelength on aggregation parameters reflecting the extent of aggregation (i.e., number of RBC per aggregate); 2. no significant effects of wavelength on parameters reflecting the time course of RBC aggregation; 3. a prominent influence of hemoglobin oxygen saturation on both extent and time-course related aggregation parameters measured at wavelengths less than 700 nm, but only on the time-course at 800 nm; and 4. the power of parameters in detecting a given alteration of RBC aggregation is affected by wavelength, in general being greater at higher wavelengths. It is recommended that light sources with wavelengths around 800 nm be used in instruments for measuring RBC aggregation via LT.

Measurement of red blood cell aggregation in disposable capillary tubes.

A new method is described in this paper that allows measurement of red blood cell (RBC) aggregation indexes in disposable glass tubes within minutes. Light transmission through the RBC suspension filled into a microhematocrit capillary at stasis is recorded during RBC aggregation; a novel method assures an initial dispersion of aggregates in the capillary. The resulting light transmittance-time data are analyzed to calculate various parameters. Measurement of erythrocyte sedimentation rate (ESR) and RBC aggregation using well established methods and the newly developed capillary tube aggregometer in blood samples with a wide range of RBC aggregation indicated significant correlations between these parameters. Additionally, light transmittance during complete disaggregation allows estimating hematocrit, thereby enabling hematocrit correction of the measured and calculated parameters. The newly developed capillary tube RBC aggregometer is suitable for use as a method to rapidly monitor disease activity and the acute phase response, especially at the point-of-care (e.g., health care facilities, physician's office) and for field studies.

Time course of electrical impedance during red blood cell aggregation in a glass tube: comparison with light transmittance.

Red blood cells (RBC) in normal human blood undergo reversible aggregation at low flow or stasis. The extent and kinetics of this phenomenon have been studied using various optical and electrical methods, yet results using such methods are not always in concordance. This study employed a horizontal glass tube in which blood flow could be established, then abruptly stopped. Normal blood and RBC suspensions with enhanced or decreased aggregation were studied. Light transmittance (LT) and electrical impedance at 100 kHz were recorded during high-shear flow and for 120 s after flow was abruptly stopped during which RBC aggregation occurs. Capacitance values were also obtained based on the imaginary part of impedance data and recorded. Various aggregation parameters were calculated, using the time course of LT, impedance, and capacitance, then compared with each other and with results from laboratory aggregometers. RBC aggregation parameters were calculated, using the time course of impedance data often failed to correlate with known changes of aggregation, even reporting aggregation for cells in nonaggregating media (i.e., RBC in buffered saline). Alternatively, RBC aggregation parameters based upon the time course of capacitance data are in general agreement with those derived from LT data and with RBC aggregation indexes, measured using commercial instruments.

Comparison of three instruments for measuring red blood cell aggregation.

The International Society for Clinical Hemorheology organized a workshop to compare three instruments for measuring RBC aggregation: LORCA, Myrenne Aggregometer and RheoScan-A. The Myrenne Aggregometer provides indices at stasis (M) and at low shear (M1), with four indices obtained with the LORCA and RheoScan-A: amplitude (AMP), half-time (T1/2), surface area (SA) above (LORCA) or below (RheoScan-A) the syllectogram, and the ratio (AI) of the area above (LORCA) or below (RheoScan-A) the syllectogram to total area (AI). Intra-assay reproducibility and biological variability were determined; also studied were RBC in diluted plasma and in 1% 500 kDa dextran, and 0.003% glutaradehyde (GA)-treated cells in plasma. All measurements were performed at 37 degrees C. Standardized difference values were used as a measure of power to detect differences. Salient results were: (1) intra-assay variations below 5% except for RheoScan-A AMP and SA; (2) biological variability greatest for T1/2 with other indices similar for the three devices; (3) all instruments detected progressive changes with plasma dilution; (4) the Myrenne and LORCA, but not the RheoScan-A, detected differences for cells in dextran; (5) GA-treatment significantly affected the LORCA (AMP, T1/2, SA, AI), the RheoScan-A (AMP, SA, AI) and the Myrenne M parameter. It is concluded that: (a) the LORCA, Myrenne and the RheoScan-A have acceptable precision and suitable power for detecting reduced aggregation due to plasma dilution; (b) greatly enhanced RBC aggregation may not be sensed by the RheoScan-A while the Myrenne M1 index may be insensitive to minor increases of cell rigidity; (c) future studies should define each instrument's useful range for detecting RBC aggregation.

Parameterization of red blood cell elongation index--shear stress curves obtained by ektacytometry.

Measurement of red blood cell (RBC) deformability by ektacytometry yields a set of elongation indexes (EI) measured at various shear stresses (SS) presented as SS-EI curves, or tabulated data. These are useful for detailed analysis, but may not be appropriate when a simple comparison of a global parameter between groups is required. Based on the characteristic shape of SS-EI curves, two approaches have been proposed to calculate the maximal RBC elongation index (EI(max)) and the shear stress required for one-half of this maximal deformation (SS(1/2)): (i) linear Lineweaver-Burke (LB) model; (ii) Streekstra-Bronkhorst (SB) model. Both approaches have specific assumptions and thus may be subject to the measurement conditions. Using RBC treated with various concentrations of glutaraldehyde (GA) and data obtained by ektacytometry, the two approaches have been compared for nine different ranges of SS between 0.6-75 Pa. Our results indicate that: (i) the sensitivity of both models can be affected by the SS range and limits employed; (ii) over the entire range of SS-data, a non-linear curve fitting approach to the LB model gave more consistent results than a linear approach; (iii) the LB method is better for detecting SS(1/2) differences between RBC treated with 0.001-0.005% glutaraldehyde (GA) and for a 40% mixture of rigid cells but is equally sensitive to SB for 10% rigid cells; and (iv) the LB and SB methods for EI(max) are equivalent for 0.001% and 0.003% GA and 40% rigid, with the SB better for 0.005% GA and the LB better for 10% rigid.

Photometric measurements of red blood cell aggregation: light transmission versus light reflectance.

Red blood cell (RBC) aggregation is the reversible and regular clumping in the presence of certain macromolecules. This is a clinically important phenomenon, being significantly enhanced in the presence of acute phase reactants (e.g., fibrinogen). Both light reflection (LR) and light transmission (LT) from or through thin layers of RBC suspensions during the process of aggregation are accepted to reflect the time course of aggregation. It has been recognized that the time courses of LR and LT might be different from each other. We aim to compare the RBC aggregation measurements based on simultaneous recordings of LR and LT. The results indicate that LR during RBC aggregation is characterized by a faster time course compared to simultaneously recorded LT. This difference in time course of LR and LT is reflected in the calculated parameters reflecting the overall extent and kinetics of RBC aggregation. Additionally, the power of parameters calculated using LR and LT time courses in detecting a given difference in aggregation are significantly different from each other. These differences should be taken into account in selecting the appropriate calculated parameters for analyzing LR or LT time courses for the assessment of RBC aggregation.

Comparison of three commercially available ektacytometers with different shearing geometries.

In December 2008, the International Society for Clinical Hemorheology organized a workshop to evaluate and compare three ektacytometer instruments for measuring deformability of red blood cells (RBC): LORCA (Laser-assisted Optical Rotational Cell Analyzer, RR Mechatronics, Hoorn, The Netherlands), Rheodyn SSD (Myrenne GmbH, Roetgen, Germany) and RheoScan-D (RheoMeditech, Seoul, Korea). Intra-assay reproducibility and biological variation were determined using normal RBC, and cells with reduced deformability (i.e., 0.001-0.02% glutaradehyde (GA), 48 degrees C heat treatment) were employed as either the only RBC present or as a sub-population. Standardized difference values were used as measure of the power to detect differences between normal and treated cells. Salient results include: (1) All instruments had intra-assay variations below 5% for shear stress (SS)>1 Pa but a sharp increase was found for Rheodyn SSD and RheoScan-D at lower SS; (2) Biological variation was similar and markedly increased for SS<3-5 Pa; (3) All instruments detected GA-treated RBC with maximal power at 1-3 Pa, the presence of 10% or 40% GA-modified cells, and the effects of heat treatment. It is concluded that the LORCA, Rheodyn SSD and RheoScan-D all have acceptable precision and power for detecting reduced RBC deformability due to GA treatment or heat treatment, and that the SS range selected for the measurement of deformability is an important determinant of an instrument's power.

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide.

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22+/-2 degrees C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10(-7)-10(-4)M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10(-3)M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.

Effects of storage duration and temperature of human blood on red cell deformability and aggregation.

Blood samples used in hemorheological studies may be stored for a period of time, the effects of storage have yet to be fully explored. This study evaluated the effects of storage temperature (i.e., 4 degrees C or 25 degrees C) and duration on RBC deformability and aggregation for blood from healthy controls and from septic patients. Our results indicate that for normal blood, RBC deformability over 0.3-50 Pa is stable up to six hours regardless of storage temperature; at eight hours there were no significant differences in EI but SS1/2 calculated via a Lineweaver-Burk method indicated impaired deformability. Storage temperature affected the stable period for RBC aggregation: the safe time was shorter at 25 degrees C whereas at 4 degrees C aggregation was stable up to 12 hours. Interestingly, blood samples from septic patients were less affected by storage. Blood can thus be stored at 25 degrees C for up to six hours for deformability studies, but should be limited to four hours for RBC aggregation; storage at 4 degrees C may prolong the storage period up to 12 hours for aggregation but not deformability measurements. Therefore, the time period between sampling and measurement should be as short as possible and reported together with results.

Effect of hemoglobin oxygenation level on red blood cell deformability and aggregation parameters.

Measurements of red blood cell (RBC) deformability and aggregation can be subject to influence by pre-analytical handling procedures, with the degree of hemoglobin oxygenation having the potential to affect the results. To examine such effects, RBC deformability and aggregation were studied before and after oxygenation or deoxygenation of human blood samples. RBC deformability was assessed using a laser-diffraction ektacytometer having Couette geometry. RBC aggregation was assessed using the same system by monitoring light backscattering after a sudden cessation of high shear; aggregation was also measured by monitoring light transmittance through RBC suspensions. RBC deformability was found to be significantly increased after equilibrating RBC with ambient air (pO2: 142.0+/-3.1 mmHg) compared to the non-oxygenated sample (pO2: 42.4+/-1.8 mmHg). In contrast, equilibration with 100% nitrogen resulted in significant impairment in RBC deformability. RBC aggregation parameters were also affected by oxygenation if measured based on light backscattering, but not if measured using light transmittance. It is thus recommended that blood samples be oxygenated by repeated exposure to ambient air prior to the measurement of hemorheological parameters.

Sampling time after tourniquet removal affects erythrocyte deformability and aggregation measurements.

Venipuncture procedures are widely thought to influence biochemical, hematological or hemorheological measurements. In line with the preparation of the new Guidelines for the standardization of hemorheological measurement, we compared various blood rheological parameters (i.e., red blood cell deformability and aggregation indices) assessed in blood samples obtained after 5, 30, 60 and 90 s following the tourniquet removal and a blood sample obtained without applying a tourniquet (control sample). A slight but significant improvement in red blood cell (RBC) deformability after the removal of tourniquet compared to the control sample was observed. RBC deformability was maximal in the samples obtained 30 s after tourniquet removal and remained slightly higher than the control in the following samples (at 60 and 90 s after tourniquet removal). The aggregation index (AI) decreased with time after tourniquet removal reaching significantly lower values than the control at 90 s after tourniquet removal. This finding was supported by a greater half time for RBC aggregation in the samples obtained 60 and 90 s after tourniquet removal. In conclusion, this study revealed that RBC deformability and aggregation might be significantly altered in the samples obtained after the application and removal of a tourniquet, as a part of the blood sampling procedure. Recommendation "remove the tourniquet at least 5 s prior to the start of blood sampling" may need to be revised.

Effect of decreased plasma cholesterol by atorvastatin treatment on erythrocyte mechanical properties.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are the most commonly used cholesterol-lowering drugs, with recent clinical trends usually aimed at achieving the lowest possible plasma cholesterol levels. Although the effects of increased plasma cholesterol have been previously reported, it is not obvious how very low plasma cholesterol levels would affect membrane composition and the deformability of red blood cells (RBC). The present study investigated the effects of hypocholesterolemia achieved by atorvastatin therapy on RBC membrane and mechanical properties in guinea pigs fed a normal diet. Two groups of animals were used (atorvastatin-treated, n=12; control n=12), and atorvastatin given orally in isotonic phosphate-buffered saline (PBS) at a dose of 20 mg/kg/day for a 21-day period. Our results indicate that the atorvastatin-treated group had significantly lower plasma total cholesterol (17.42+/-1.70 mg/dl), low-density lipoprotein cholesterol (5.25+/-2.22 mg/dl) and triglycerides (42.60+/-3.78 mg/dl) than the control group (34.08+/-1.72, 21.17+/-1.41 and 60.64+/-2.43 mg/dl, respectively). In addition, membrane cholesterol content was lower (p<0.0001) and phospholipid content higher (p<0.0001) in the atorvastatin-treated group, thus decreasing the cholesterol to phospholipid ratio; a significant enhancement in sodium-potassium-ATPase activity also occurred. However, in spite the marked changes of plasma and RBC membrane composition, there was no change of RBC deformability. Note that although our results indicate no adverse rheological alterations, extension of our findings to humans requires caution.

Effects of red blood cell aggregation on myocardial hematocrit gradient using two approaches to increase aggregation.

The normal transmyocardial tissue hematocrit distribution (i.e., subepicardial greater than subendocardial) is known to be affected by red blood cell (RBC) aggregation. Prior studies employing the use of infused large macromolecules to increase erythrocyte aggregation are complicated by both increased plasma viscosity and dilution of plasma. Using a new technique to specifically alter the aggregation behavior by covalent attachment of Pluronic F-98 to the surface of the RBC, we have determined the effects of only enhanced aggregation (i.e., Pluronic F-98-coated RBCs) versus enhanced aggregation with increased plasma viscosity (i.e., an addition of 500 kDa dextran) on myocardial tissue hematocrit in rapidly frozen guinea pig hearts. Although both approaches equally increased aggregation, tissue hematocrit profiles differed markedly: 1) when Pluronic F-98-coated cells were used, the normal transmyocardial gradient was abolished, and 2) when dextran was added, the hematocrit remained at subepicardial levels for about one-half the thickness of the myocardium and then rapidly decreased to the control level in the subendocardial layer. Our results indicate that myocardial hematocrit profiles are sensitive to both RBC aggregation and to changes of plasma viscosity associated with increased RBC aggregation. Furthermore, they suggest the need for additional studies to explore the mechanisms affecting RBC distribution in three-dimensional vascular beds.