Elevated plasma dopamine beta hydroxylase activity in rats with neurogenic hypertension.

Increased plasma dopamine beta hydroxylase, DBH, activity has been cited as evidence of increased sympathetic function in essential hypertension. Here-to-fore, experimental hypertension in animals has been associated with normal plasma DBH activity. This study shows that rats with neurogenic hypertension, induced by sinoaortic denervation, SAD, have elevated DBH activity; the mean increase in plasma DBH measured 3 days to 11 weeks after operation was 74% higher in the SAD group than in the sham-operated, control group. DBH activity showed a positive correlation with arterial pressure. Mesentery DBH activity was inversely related to plasma enzyme activity in SAD rats, indicating sympathetic nerve terminals in mesentery are a source of plasma DBH. We conclude that plasma DBH activity is an index of increased sympathetic function since it is consistently elevated in rats with neurogenic hypertension resulting from sustained central activation of the sympathetic nervous system.     

Plasma Renin Activity and Blood Pressure in 89 Patients Receiving Maintenance Haemodialysis Therapy

Blood pressure, plasma renin activity, plasma sodium concentration, plasma potassium concentration, dietary sodium intake, and duration of dialysis have been measured under standard conditions in 89 patients on maintenance haemodialysis. No significant relation was found between plasma renin activity and blood pressure. Statistically significant correlations were found between plasma renin activity and plasma sodium concentration and between plasma renin activity and dietary sodium intake.Only one patient was found to have uncontrollable hypertension associated with a markedly raised plasma renin activity. Reasons are given for not performing bilateral nephrectomy in this patient. We believe the low incidence of uncontrollable hypertension and hyperreninaemia in our patients to be due to their slow introduction to haemodialysis, thus preventing violent swings in body weight, blood pressure, and renin secretion.Although plasma renin activity did fall with duration of dialysis, all 15 patients who have been on maintenance dialysis for longer than five years have normal levels.     

The measurement of lysosomal phospholipase A2 activity in plasma

A deficiency of lysosomal phospholipase A2 (LPLA2) causes macrophage-associated phospholipidosis, suggesting that the enzyme is important in the lipid catabolism. Because LPLA2 is secreted by macrophages, extracellular LPLA2 activity may potentially reflect a change in macrophage activation. In this report, the detection of LPLA2 activity in plasma was established by the measurement of the transacylase activity of LPLA2 under acidic conditions. No transacylase activity of LPLA2 was detected in normal human plasma when the plasma was incubated with liposomes consisting of 1,2-dioleoylphosphatidylcholine/sulfatide/N-acetylsphingosine (NAS) at pH 4.5. However, the transacylase activity in the plasma was detected when liposomes consisting of 1,2-dioleoylphosphatidylglycerol/NAS were used as a substrate. To establish the specificity of the assay, ceramide transacylase activity was detected in the plasma of wild-type mice. By contrast, the plasma obtained from LPLA2-knockout mice had no measurable transacylase activity under the same conditions. The enzymatic activity of recombinant LPLA2 was inhibited by treatment with methylarachidonylfluorophosphonate. The inhibitor also suppressed the transacylase activity observed in both normal human and wild-type mouse plasma, establishing that the transacylase activity observed in plasma is due to LPLA2. Plasma LPLA2 activity may be a useful bioassay marker for the identification of LPLA2-related disorders.     

Sodium outflow rate through lymphocyte cell membranes, serum levels of sodium, potassium, aldosterone, total catecholamines, 6-keto- PGF2alpha and plasma renin activity in patients with primary arterial hypertension treated with captopril]

Sodium ions outflow rate through lymphocyte membranes, serum sodium, potassium, aldosterone, total catecholamines and 6-keto-PGE alpha levels, and plasma renin activity were studied in patients with mild hypertension associated with low and hugh plasma renin activity treated with captopril in a single dose of 12.3 mg and after the treatment with daily doses of 12.5 mg and 25 mg for 3 days. It was found, that captopril in hypertensive patients with high plasma renin activity decreases both systolic and diastolic blood pressure, decelerates heart rate, and decreases serum total catecholamines and plasma renin activity. Sodium ions outflow rate and serum sodium, potassium, aldosterone, and 6-keto-PGE alpha remain unchanged. Captopril in hypertensive patients with low plasma renin activity. The remaining parameters are unchanged. Moreover, it was noted that serum 6-keto-PGE alpha levels are lower in hypertensive patients with low plasma renin activity.     

Response of plasma and red cell glutathione reductase of several mammalian species and domestic chickens to flavin adenine dinucleotide.

Glutathione reductase (GR) activity was determined in the plasma and red cells from a number of species (human, horse, cattle, sheep, chicken, rabbit, rat). The species with the highest plasma activity (horse and cattle) had the lowest red cell activity. Unlike red cell GR, plasma GR was not significantly stimulated by the addition of flavin adenine dinucleotide (FAD). In species with high plasma GR activity the determination of whole blood FAD activity coefficients may not be a good index of riboflavin status.     

Differential response of the NADH oxidase of plasma membranes of rat liver and hepatoma and HeLa cells to thiol reagents

NADH oxidase activity of plasma membranes from rat hepatoma and HeLa cells responded to thiol reagents in a manner different from that of plasma membranes of liver. Specifically, the NADH oxidase activity of plasma membranes of HeLa cells was inhibited by submicromolar concentrations of the thiol reagentsp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), or 5,5-dithiobis-(2-nitrophenylbenzoic acid) (DTNB), whereas that of the rat liver plasma membranes was unaffected or stimulated over a wide range of concentrations extending into the millimolar range. With some hepatoma preparations, the NADH oxidase activity of hepatoma plasma membranes was stimulated rather than inhibited by PCMB, whereas with all preparations of hepatoma plasma membranes, NEM and DTNB stimulated the activity. In contrast, NADH oxidase activity of rat liver plasma membrane was largely unaffected over the same range of PCMB concentrations that either stimulated or inhibited with rat hepatoma or HeLa cell plasma membranes. Dithiothreitol and glutathione stimulated NADH oxidase activity of plasma membranes of rat liver and hepatoma but inhibited that of HeLa plasma membranes. The findings demonstrate a difference between the NADH oxidase activity of normal rat liver plasma membranes of rat hepatoma and HeLa cell plasma membranes in addition to the differential response to growth factors and hormones reported previously (Brunoet al., 1992). Results are consistent with a structural modification of a NADH oxidase activity involving thiol groups present in plasma membranes of rat hepatoma and HeLa cells but absent or inaccessible with plasma membranes of rat liver.     

Biochemical and genetic studies of plasma and leukocyte α-L-fucosidase

Deficiency of alpha-L-fucosidase in plasma and leukocytes has been demonstrated in three patients affected with fucosidosis. The measurement of plasma fucosidase activity alone is of little diagnostic value. Several normal individuals were found to have extremely low plasma alpha-L-fucosidase activity, but normal activity in leukocyte preparations. The low plasma enzyme activity exhibited by clinically normal individuals appears to be an inherited characteristic. The plasma enzyme was found to be different from that of leukocytes in terms of electrophoretic mobility.     

Plasma myeloperoxidase activity as a criterion of therapeutic effectiveness for patients with cardiovascular diseases

A significant increase in the myeloperoxidase (MPO) activity has been found in plasma of patients with stable angina and with acute coronary syndrome (ACS) in comparison with the control group. MPO concentration was significantly increased in plasma of ACS patients. Reduced MPO activity in the treated ACS patients correlated with a favorable outcome of the disease. Generally, changes in plasma MPO concentration coincided with changes in lactoferrin concentration thus confirming the role of neutrophil degranulation in the increase of plasma concentrations of these proteins. The increase in MPO activity was obviously determined by modification of the MPO protein caused by reactive oxygen species and halogen in the molar ratio of 1: 25 and 1: 50. The decrease in plasma MPO activity may be associated with increased plasma concentrations of the physiological inhibitor of its activity, ceruloplasmin, and also with modification of the MPO protein with reactive oxygen species and halogen at their molar ratio of 1: 100 and higher. Thus, MPO activity may be used for evaluation of effectiveness of the treatment of cardiovascular diseases.     

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 μl of plasma and 240 μl of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 μM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.     

Corticotropin releasing factor (CRF) activity in rat plasma.

The ACTH-releasing activity of hypothalamic extract and rat plasma was examined with the dispersed rat pituitary cell technique of Swallow and Sayer (8). Although both plasma and serum caused ACTH release which was dose-related, stress did not enhance the ACTH releasing activity. Furthermore, separation studies of plasma using ultrafiltration and gel separation suggest that the CRF activity in plasma is associated with molecules of a molecular weight greater than 15,000.     

An investigation of carbonic anhydrase activity in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate (Raja rhina), and spotted ratfish (Hydrolagus colliei)

Separated plasma and whole blood non-bicarbonate buffering capacities, together with plasma and gill carbonic anhydrase activities and endogenous plasma carbonic anhydrase inhibitor activity were investigated in three species of fish: the brown bullhead (Ameirus nebulosus), a teleost; the longnose skate (Raja rhina), an elasmobranch; and the spotted ratfish (Hydrolagus colliei), a chimaeran. The objective was to test the hypothesis that species possessing gill membrane-bound carbonic anhydrase and/or plasma carbonic anhydrase activity would also exhibit high plasma nonbicarbonate buffering capacity relative to whole blood non-bicarbonate buffering capacity and would lack an endogenous plasma carbonic anhydrase inhibitor. Separated plasma non-bicarbonate buffering capacity constituted > or = 40% of whole-blood buffering in all three species. In addition, all species lacked an endogenous plasma carbonic anhydrase inhibitor. Separated plasma from skate and ratfish contained carbonic anhydrase activity, whereas bullhead plasma did not. Examination of the subcellular distribution and characteristics of branchial carbonic anhydrase activity revealed that the majority of branchial carbonic anhydrase activity originated from the cytoplasmic fraction in all species, with only 3-5% being associated with a microsomal fraction. The microsomal carbonic anhydrase activity of bullhead and ratfish was significantly reduced by washing, indicating the presence of carbonic anhydrase activity that was not integrally associated with the membrane pellet, microsomal carbonic anhydrase activity in skate was unaffected by washing. In addition, microsomal carbonic anhydrase activity from skate and ratfish but not bullhead gills was released to a significant extent from its membrane association by treatment with phosphatidylinositol-specific phospholipase C. The results obtained for skate are consistent with published data for dogfish, suggesting that the possession of branchial membrane-bound carbonic anhydrase activity may be a generalised elasmobranch characteristic. Ratfish, which also belong to the class Chondrichthyes, exhibited a similar pattern. Unlike skate and ratfish, bullhead exhibited high plasma non-bicarbonate buffering capacity and lacked an endogenous carbonic anhydrase inhibitor in the absence of plasma and gill membrane-bound carbonic anhydrase activities.     

Reduction of cyclic AMP phosphodiesterase activity of several subcellular fractions of rat brain after pretreatment with phospholipase C

Cyclic AMP phosphodiesterase (PDE) activity was assayed in the plasma membrane, mitochondrial and microsomal fractions of rat brain. The specific activity of the enzyme was highest in the plasma membrane fraction followed by mitochondrial and then the microsomal fraction. Phosphodiesterase activity of all three fractions was reduced after pretreatment with lecithinase C (PCase) from Clostridium perfringens but less markedly affected by the pretreatment with sphingomyelinase (SMase) from human placenta. The PDE activity of the plasma membrane fraction was more sensitive to PCase treatment compared with the other two particulate fractions, which showed only a slight loss of activity. Temperature seemed to affect PDE activity of the plasma membrane. The enzyme was quite stable at 30 degrees C but its activity dropped by approximately 46% at 37 degrees C after 90 min of incubation. Pretreatment of the plasma membrane at 30 degrees C with PCase at a concentration of more than 5 U caused a marked loss of PDE activity and the decrease in activity reached a plateau at concentrations above 10 U.     

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.     

Modifications of arterial pressure and plasma renin: their effects on the norepinephrine content of hypothalamus and medulla oblongata.

The effects of the bilateral nephrectomy and acute hypotension caused by the cava vein ligature on the norepinephrine (NE) concentration in hypothalamus and medulla oblongata and on the plasma renin activity were studied in male rats. NE increased and plasma renin activity decreased in hypothalamus in 24 h nephrectomized rats with or without the ligatures of the cava vein. NE also decreased in medulla of groups with the ligatures only. The mean arterial pressure, did not correlate with the NE or plasma renin activity levels. The modifications of the NE in the central nervous system showed an inverse relationship with plasma renin activity and this, could be due to changes in the NE uptake and/or release caused by the plasma renin activity alterations. NE modifications do not seem to be caused directly through reflex of the arterial pressure.     

Elevated plasma homocysteine elicits an increase in antioxidant enzyme activity

Elevated plasma homocysteine is considered to be a risk factor for cardiovascular disease. The mechanisms for this effect are not fully understood but there is some evidence for a role for reactive oxygen species (ROS). This study was conducted to explore the effects of elevated plasma total homocysteine (tHcy) concentration on activity of antioxidant enzymes in the circulation. The study group consisted of 10 patients with inherited defects of homocysteine metabolism, from whom 41 blood samples were collected over a period of six months. Blood samples were also collected from 13 of their obligate heterozygous parents. For data analysis samples were classified as those with plasma tHcy < 20 μM or ≥ 20 μM. The activity of erythrocyte superoxide dismutase (SOD) and plasma glutathione peroxidase (GSHPx) was elevated in samples with plasma tHcy > 20 μM. Moreover, a significant correlation was demonstrated between plasma GSHPx activity, plasma glutathione peroxidase protein and plasma tHcy. In vitro studies confirmed that this observation was not due to a simple chemical enhancement of enzyme activity. Homocysteine protected GSHPx from loss of activity following incubation at 37°C. A similar effect was seen with another thiol-containing amino acid, cysteine. Results suggest that elevated plasma tHcy represents an oxidative stress, resulting in an adaptive increase in activity of antioxidant enzymes in the circulation.     

Elevated plasma homocysteine elicits an increase in antioxidant enzyme activity

Elevated plasma homocysteine is considered to be a risk factor for cardiovascular disease. The mechanisms for this effect are not fully understood but there is some evidence for a role for reactive oxygen species (ROS). This study was conducted to explore the effects of elevated plasma total homocysteine (tHcy) concentration on activity of antioxidant enzymes in the circulation. The study group consisted of 10 patients with inherited defects of homocysteine metabolism, from whom 41 blood samples were collected over a period of six months. Blood samples were also collected from 13 of their obligate heterozygous parents. For data analysis samples were classified as those with plasma tHcy < 20 microM or > 20 microM. The activity of erythrocyte superoxide dismutase (SOD) and plasma glutathione peroxidase (GSHPx) was elevated in samples with plasma tHcy > 20 microM. Moreover, a significant correlation was demonstrated between plasma GSHPx activity, plasma glutathione peroxidase protein and plasma tHcy. III vitro studies confirmed that this observation was not due to a simple chemical enhancement of enzyme activity. Homocysteine protected GSHPx from loss of activity following incubation at 37 degrees C. A similar effect was seen with another thiol-containing amino acid, cysteine. Results suggest that elevated plasma tHcy represents an oxidative stress, resulting in an adaptive increase in activity of antioxidant enzymes in the circulation.     

Measuring plasminogen activator inhibitor activity in plasma by two enzymatic assays

We compared two methods that measure plasminogen activator inhibitor (PAI) activity in plasma based on the ability of PAI to inhibit tissue plasminogen activator (tPA) or urokinase (uPA) in order to determine which method most accurately measures plasma PAI activity after stressors, like hemorrhage. Plasma PAI activity was significantly elevated after hemorrhage in both assays. Using standard curves derived from rhPAI-1, we found that the tPA-PAI assay was more sensitive than the uPA-PAI assay. However, we measured a 10-fold difference in PAI activity as measured between assays, suggesting that some endogenous plasma constituents (tPA, uPA, plasminogen or plasmin) may interfere with the accurate determination of PAI activity. Increasing the amount of plasma in each assay led to a progressive increase in PAI activity. However, removing either tPA or plasminogen from the tPA-PAI assay unmasked the presence of some endogenous tPA and plasminogen. Furthermore, increasing plasma volume in either assay increases measured plasma tPA, but not uPA. Finally, plasma tPA is elevated after hemorrhage, whereas plasma uPA is not. These results suggest that endogenous tPA and plasminogen may interfere with the measurement of plasma PAI activity in the tPA-PAI assay after hemorrhage or other stresses. The uPA-PAI assay does not have this confounding problem because endogenous uPA does not interfere with the assay, nor does it rise during hemorrhage.     

Disappearance and effects of exogenous lipid transfer activity in rats

These studies were performed to determine the role of plasma lipid transfer activity in the regulation of plasma total and lipoprotein cholesterol in vivo. Partially purified human lipid transfer activity was injected into rats at a level similar to that of normal rabbit plasma, d greater than 1.21. The disappearance of exogenous lipid transfer activity from rat plasma was biphasic, with a 70% loss within 6 h. The remaining 30% was lost with a half-time of about 14 h. In the rat, short-term exposure (6 h) to high levels of lipid transfer activity resulted in a net transfer of cholesteryl esters from high density to d less than 1.019 lipoproteins, without affecting plasma total cholesterol. However, the lipid transfer activity-induced changes in lipoprotein cholesterol were not evident after 24 h, despite the fact that the lipid transfer activity of rat plasma d greater than 1.21 was similar to that of human plasma d greater than 1.21 during the preceding 18 h.     

Effect of plasma from hypertensive patients on contractile response of vascular smooth muscle from normotensive rat

This study examines whether incubation with plasma from essential hypertensive patients increases the contractile activity of vascular smooth muscle from rats in response to noradrenaline (NA) and potassium (K+). Plasma samples were obtained from age- and sex-matched essential hypertensive patients and normotensive people. Vascular strips were prepared from aorta and portal veins of normotensive rats and placed in physiological solution in muscle baths for measurement of mechanical response. Aortic strips exposed to hypertensive plasma showed increased responsiveness to NA compared with normotensive plasma, but K+ caused an opposite effect. Portal vein exposed to normotensive or hypertensive plasma did not produce any response to NA, but the responsiveness produced in the presence of normotensive plasma to K+ was higher than that of hypertensive plasma. Portal vein exposed to normotensive plasma or hypertensive plasma showed a dose-dependent increase in the spontaneous activity up to 50% concentration of the plasma samples, but further increase in the concentration of plasma inhibited the spontaneous activity. Spontaneous activity at any given concentration of hypertensive plasma was significantly higher than that of normotensive plasma. The spontaneous activity in the presence of heated or unheated normotensive plasma or unheated normotensive serum was not significantly different from each other. These results indicate that the plasma factor from hypertensive patients, which alters the reactivity of vascular smooth muscle from normotensive rat, is present in the serum fraction and is not heat sensitive.     

Human plasma glutathione oxidation in normal and pathological conditions

Reduced glutathione added to human plasma disappears rapidly, and it is concomitantly recovered in its oxidized form. This oxidation is not due to the plasma metal content since it is not inhibited by EDTA or by passage of plasma through Chelex columns. Furthermore, this oxidation is not due to the peroxidase activity of glutathione S-transferases, which are usually undetectable in normal human serum, and it does not correlate with the amount of plasma glutathione peroxidase. A significant increase in the rate of glutathione oxidation was observed in plasma of patients with increased gamma-glutamyltranspeptidase activity. It is concluded that the side-oxidase activity of gamma-glutamyltransferase is responsible for the oxidation of glutathione in human plasma.