To examine the relationship between arachidonic acid metabolism and sepsis, we determined the blood concentration of platelet-activating factor acetylhydrolase (PAFAH) and of arachidonic acid cascade substances, leukotriene B4 (LTB4), thromboxane B2 (TXB2), and 6-keto-prostaglandin Fla (PGF1a), in patients with clinical sepsis. Blood concentrations of PAFAH, LTB4, TXB2 and PGF1a at the time of the initial diagnosis of sepsis were 23.9 +/- 9.7 nmol/min/mL, 90.4 +/- 31.5 pg/mL, 55.3 +/- 44.1 pg/mL and 22.3 +/- 11.9 pg/mL, respectively. There was a significant correlation between the blood PAFAH and LTB4 concentration (r = 0.520, p = 0.0033) and the diagnosis of sepsis. There also was a significant correlation between the blood PAFAH and TXB2 concentration (r = 0.460, p = 0.0311). There was no correlation between the blood PAFAH and PGF1a concentrations. Elevated LTB4 and TXB2 concentrations showed a significant correlation with the PAFAH concentration in patients with sepsis, suggesting a direct relationship between these substances.
In rats intoxicated with doxorubicin, vasomotor responses were evaluated 12 hr after the last dosage. In rats pretreated with doxorubicin, hypertensive responses to L-norepinephrine and L-epinephrine, and hypertension by occlusion of both common carotid arteries were significantly (p < 0.05) reduced when compared with controls. Doxorubicin pretreatment also significantly reduced the arterial hypotension due to L-isoprenaline. In rats intoxicated with doxorubicin, pretreatment with L-sulpiride (12.5 to 50 mg/Kg/day for 30 days in drinking water ad libitum) did not modify the effects of doxorubicin on vasomotor reactivity. In contrast, pretreatment with amitriptyline (12.5 to 50 mg/Kg/day in drinking water ad libitum for 30 days) potentiated the inhibitory effects of doxorubicin on vasomotor responses. In conclusion, our research shows that doxorubicin intoxication induces a significant reduction of alpha- and beta-adrenergic reactivity and of baroreceptor activity.
As part of our ongoing work to develop the new non-isotopic assay method carbonyl metalloimmunoassay (CMIA), whose efficacy has already been proven in the laboratory for phenobarbital and cortisol, we here present the steps involved in establishing CMIA of 5,5 diphenylhydantoin (DPH), one of the most commonly used antiepileptic medications. First, anti-DPH antibodies were obtained by injection of the immunogen DPH-3-valerate-BSA into rabbits. The titer value and specificity of these antibodies were examined by RIA using [14C]-DPH as tracer, and an antibody batch selected for its high titer value and good specificity for metabolites of DPH and other antiepileptic drugs. Next the organometallic complex Cr(CO)3-DPH, chosen as the CMIA tracer, was synthesized and shown to conserve a high recognition value for anti-DPH antibodies (CR = 200%). Isopropyl ether was selected as the best organic solvent for use in separating the free and bound fractions of the tracer. Employing the Cr(CO)3-DPH complex as tracer and FT-IR spectroscopy as the detection method, we were able to obtain a titration curve by CMIA using an amount of tracer identical to that used in RIA. The titer value obtained in CMIA is approximately twice that obtained by RIA. These results demonstrate the feasibility of DPH assay by the CMIA method.
The effect of acute hyperinsulinemia on plasma lipoprotein a [Lp(a)] concentration in 25 patients with non-insulin dependent diabetes mellitus (NIDDM) and in 10 healthy subjects was examined. Insulin was infused into the subjects for 2 hours while baseline plasma glucose concentrations were maintained. Plasma Lp(a) levels showed a small but significant mean (+/- SD) increase in NIDDM patients (12 +/- 26 U/l, p < 0.01) but did not vary significantly in healthy subjects (3 +/- 15 U/l) when insulin was infused. There was considerable individual variation (-35 to 98 U/l) in the response of Lp(a) to acute hyperinsulinemia and the response was correlated significantly with baseline Lp(a) levels (r = 0.399, p < 0.05). These data suggest that acute hyperinsulinemia at constant baseline glucose levels may raise plasma Lp(a) levels in NIDDM patients more particularly in those with high initial Lp(a) concentrations.
A novel reductive metabolism of shogaol [1-(4'-hydroxy-3'-methoxyphenyl)-deca-4-ene-3-one], a major pungent and pharmacologically active principle of ginger, was investigated in rat liver in vitro. The ethyl acetate extractable metabolites formed by incubation of this alpha,beta-unsaturated ketone with rat liver 12,000 x g supernatant fortified with NADPH-generating system were analyzed by high performance chromatography and gas chromatography/mass spectrometry. In addition to the saturated ketone and reduced alcohol metabolites, an allyl alcohol, 1-(4'-hydroxy-3'-methoxyphenyl)-deca-4-ene-3-ol, was identified as a new metabolite of shogaol. Likewise, dehydroparadol [1-(4'-hydroxy-3'-methoxyphenyl)-deca-1-ene-3-one], a non-pungent analog of shogaol, was also reduced to the corresponding allyl alcohol by the postmitochondrial fraction of rat kidney in the presence of NADPH-generating system.
The effect of hydrogen peroxide on metabolism of L-arginine to L-citrulline in the biosynthesis of nitric oxide was investigated in bovine aortic endothelial cells. Addition of hydrogen peroxide (1-10 mM) to endothelial cells increased L-citrulline formation from L-arginine in a dose-dependent manner. Stimulation of L-citrulline formation was prevented by catalase (20 micrograms/ml), a hydrogen peroxide scavenger, but not by superoxide dismutase (SOD, 100 micrograms/ml), a superoxide anion scavenger. Pretreatment with NG-nitro-L-arginine (10(-5) M) and NG-methyl-L-arginine (10(-4) M), potent inhibitors of nitric oxide synthase, also inhibited L-citrulline formation induced by hydrogen peroxide. Moreover, hydrogen peroxide increased intracellular Ca2+ concentrations, and the removal of extracellular Ca2+ reduced the hydrogen peroxide-induced formation of L-citrulline. These findings show that hydrogen peroxide increases the intracellular Ca2+ concentration and stimulates the formation of L-citrulline from L-arginine coupled with nitric oxide synthesis in cultured endothelial cells.
Species-specific differences in the inflammatory response, specifically with regard to mast cells, have been proposed to explain the phenotypic variation among dystrophin-deficient humans, and mdx mice (Gorospe et al., 1994). To test this hypothesis we have intramuscularly injected a mast cell secretogogue into both dystrophin-negative mdr and dystrophin-positive normal mice. Mast cell activity was determined by measuring the activity of mast cell tryptase, while creatine kinase activity was used to determine the course of muscle damage in vivo. Area of damage around the injection site was measured at autopsy, and used as an indication of relative sensitivity to the secretogogue effect of compound 48/80. Mdr mice exhibited more damage in response to intramuscular injection than normal control mice. In addition, mdx mice showed a substantial increase in plasma tryptase activity, followed by a large increase in muscle creatine kinase activity. On the other hand, dystrophin-positive normal controls injected with 48/80 liberated little CK or tryptase activity. These results are consistent with the hypothesis that species-specific differences in mast cell activity, or sensitivity to mast cell products could account for the variation in pathology seen in dystrophin-deficient animals.
Contractile responses to norepinephrine (noradrenalin, NE 10(-5)M) in the canine saphenous vein (SV) are significantly, although slightly, reduced (14%) when induced in a physiological medium depleted of calcium for 1 hour (+ EGTA). In contrast, they are inhibited by about 75% after 24 hr in calcium free physiological saline solution (P.S.S.). ED(50) of norepinephrine in 1-hr calcium-free medium and in normal Ca++ P.S.S. are 6 x 10(-7)M and 4.2 x 10(-7)M respectively.Two blockers of extracellular calcium entry have also been cited as inhibitors of intracellular calcium pool refilling. At concentrations of 10(-6)M, 10(-5)M and 10(-4)M, diltiazem and nicardipine inhibit the norepinephrine-induced contractions (NIC) in a concentration-dependent manner. At 10(-4)M, the two calcium blockers inhibit the NIC by 70% and by 72% respectively in Ca++ free (+ EGTA) P.S.S.Nifedipine and verapamil only begin to significantly inhibit NIC in Ca++ free (+EGTA) P.S.S at concentrations equal to or greater than 10(-5)M. At 10(-4)M concentration, control inhibition in Ca++ free P.S.S was observed as 60% and 49%, respectively. Contrary to the other 3 calcium antagonists tested, diltiazem antagonises NIC significantly less in calcium-containing medium (45%) than in calcium-free medium (72%). Procaine at a concentration of 10(-3)M, described as sufficient to totally inhibit calcium release from its intracellular storage sites, only inhibits NIC by 52% in calcium free (+ EGTA) P.S.S.These results are consistent with the following conclusions : i) in the canine saphenous vein (SV), NIC is mainly mediated by calcium mobilization from its intracellular storage sites; ii) the calcium antagonists tested here and procaine are unable to totally inhibit, even at high concentrations, the contractions induced via intracellular calcium release; this characteristic is nonsignificant for nifedipine and verapamil at low concentrations (10(-6)M). iii) verapamil and nifedipine, like diltiazem and nicardipine at high concentrations, may not only possess the characteristics of extracellular calcium entry blockers, but also that of partial antagonist of NIC via non specific mechanisms; iv) diltiazem may relax the vascular smooth muscle of SV, not only by the above two properties, but also through another mechanism yet unknown; v) partial persistence of NIC on the SV under conditions of short or long extracellular calcium depletion may be due to a mechanism of intracellular Ca++ recycling, the smooth muscle cell partially retaining its intracellular Ca++.
To elucidate the relationship between active oxygen and interleukin 8 (IL-8) in patients with septic adult respiratory distress syndrome (ARDS), we determined the serum levels of alpha-tocopherol, which has an antioxidant action, and IL-8 in seven patients with this disease. Serum alpha-tocopherol and IL-8 levels determined at the time of diagnosis of septic ARDS were 0.97 +/- 0.36 mg/dl and 0.98 +/- 0.99 ng/ml, respectively. A significant correlation was found between serum alpha-tocopherol level and IL-8 level (r = -0.758, p = 0.0473). These findings suggest that IL-8 activates neutrophils, which produce active oxygen.
Species-specific differences in the inflammatory response, specifically with regard to mast cells, have been proposed to explain the phenotypic variation among dystrophin-deficient humans, and mdx mice (Gorospe et al., 1994). To test this hypothesis we have intramuscularly injected a mast cell secretogogue into both dystrophin-negative mdx and dystrophin-positive normal mice. Mast cell activity was determined by measuring the activity of mast cell tryptase, while creatine kinase activity was used to determine the course of muscle damage in vivo. Area of damage around the injection site was measured at autopsy, and used as an indication of relative sensitivity to the secretogogue effect of compound 48/80. Mdx mice exhibited more damage in response to intramuscular injection than normal control mice. In addition, mdx mice showed a substantial increase in plasma tryptase activity, followed by a large increase in muscle creatine kinase activity. On the other hand, dystrophin-positive normal controls injected with 48/80 liberated little CK or tryptase activity. These results are consistent with the hypothesis that species-specific differences in mast cell activity, or sensitivity to mast cell products could account for the variation in pathology seen in dystrophin-deficient animals.
The activities (C3a, C4a, C5a) and concentrations (CH50, C3, C4, C5) of serum complement were measured to evaluate the involvement of complement in sepsis. We studied 27 patients with sepsis who were divided into the survivors (Group 1) and the nonsurvivors (Group 2). The levels of C3a, C4a, and C5a were all significantly higher in Group 2 than in Group 1 and closely reflected the severity of sepsis. Levels of C3 and C4 were significantly lower in Group 2 than in Group 1, presumably because these components were consumed as a result of activation of both the alternative and classical pathways. The levels of CH50 and C5 did not differ significantly between the two groups. These findings suggest that complement is closely involved in the exacerbation of the condition of septic patients, and that the measurement of complement activity is useful for evaluating the severity of sepsis.
The influence of stevioside, the sweet glycoside of Stevia rebaudiana leaves, on the glycogen levels of fasted rats was investigated. In one set of experiments, single doses of stevioside (200 mumol) or steviol (200 mumol) were given orally to 24-hours fasted rats, either alone or simultaneously with fructose. Under these conditions both stevioside and steviol increased the initial glycogen deposition in the liver. In another set of experiments, stevioside was given to the rats in the drinking water at the beginning of the fasting periods (5:00 p.m.) of 24 and 48 hours. Two different concentrations were given, 1.0 and 2.0 mM. Increased hepatic glycogen levels were found at 48 hours with stevioside (1.0 mM) and at 24 hours with stevioside (2.0 mM). Steviol had no effect on hepatic glycogen levels when given in the drinking water. It can be concluded that stevioside exerts a stimulatory action on hepatic glycogen synthesis under gluconeogenic conditions.
Experiments were performed on normotensive rats exposed to vitamin D deficient and control diets from the 22nd to the 180th day of age. In 60-120- and 180-day-old rats. The following parameters were evaluated: a) The vasomotor responses elicited by receptor agonists in the absence and in the presence of the respective antagonists [L-norepinephrine (NE) before and 5 min after prazosin; L-isoprenaline (I) before and 5 min after DL-propranolol; L-dopamine (DA) before and 5 min after L-sulpiride or SCH 23390 or chlorpromazine; acetylcholine (Ach) before and 5 min after atropine; histamine (H) before and after chlorpheniramine; 5-hydroxytryptamine (5-HT) before and 5 min after methysergide or ketanserin]; by carotid-sinus baroreceptor stimulation (CO) before and 5 min after hexamethonium, and by electrical stimulation of the vagus peripheral head (V) before and after atropine; b) Reflex tachycardia elicited by bilateral carotid occlusion (CO) (for 40 sec) and by sodium nitroprusside; c) Catecholamine (norepinephrine, epinephrine) and arginine-vasopressin plasma levels; d) Cholesterol, triglyceride and electrolyte (Na+, K+, Cl-, Ca2+) serum levels. Our results showed that vitamin D deficient diets induced a decrease in pressor responses to NE and CO, and an increase in hypotensive responses to I, DA, Ach, H, 5-HT and V. Changes of arterial blood pressure, heart rate, catecholamine and arginine-vasopressin plasma levels were not observed. Cholesterol, triglyceride and electrolyte (Na+, K+, Cl-) serum levels were not modified, while Ca2+ serum levels decreased. In conclusion, our data suggest that vitamin D depletion can induce changes of pressor and depressor vasomotor responses and suppose a direct role for vitamin D in regulating vasomotor reactivity.
In human fibroblast cultures TPA increased IL-6 and IL-8 production. This was reduced by vitamin D3 metabolites and analogs. The two analogs employed: 1,25 (OH)2-22 (E)-dehydro-24-monohomo vitamin D3 (Compound A) and 1,25 (OH)2 -22 (E)-dehydro-24 dihomo-vitamin D3 (Compound B) may be useful in the therapy of pathologic proliferative disorders including psoriasis, particularly since they are less toxic and have less effect on calcium metabolism than vitamin D3.
Two new analogs of 1,25 dihydroxy vitamin D3 (calcitriol, DHCC), the active metabolite of vitamin D3 (cholecalciferol (D3) are less active on calcium metabolism and less toxic than (DHCC). They had increased differentiation-inducing activity towards normal on human myeloblastic leukemia cell line HL-60 and on histiocytic lymphoma cell line U-937 consisting of monoblastoid cells. 1,2 tetradeconyl phorbol 1, 3 acetate (TPA) was used as a positive control in these experiments.
The effects of intramuscular injections of minute amounts of polymyxin B were studied in 42 patients with endotoxemia. Plasma endotoxin was measured by means of an endotoxin-specific Endospecy test (Seikagaku Corp., Tokyo, Japan) after pretreatment of the plasma with a new perchloric acid method that we developed. The normal value of plasma endotoxin is less than 9.8 pg/mL. Polymyxin B was administered at a dose of 12,500 U every 6 hours. Plasma endotoxin rapidly decreased to the normal range in 40 of the 42 patients. Body temperature fell significantly. APACHE II scores were also significantly improved. Tumor necrosis factor-alpha and interleukin-6 (IL-6) decreased in survivors, while tending to persist in high values in patients who died. No side effects were observed in any of the patients. In conclusion, intramuscular injections of small doses of polymyxin B were useful in the treatment of endotoxemia.
To examine whether the dosage of a platelet-suppressive agent at which an antithrombotic effect is adequate and bleeding tendency is not increased can be found, the antithrombotic effects, antiplatelet effects and bleeding times of ticlopidine and aspirin were investigated in the rat experimental thrombus formation model. Thrombus formation was determined by measuring the change in wet weight of a silk thread placed in a carotid arteriovenous shunt. Ticlopidine inhibited thrombus formation and platelet aggregation at rather low doses (50-100 mg/kg) without prolonging bleeding time. However, aspirin did not inhibit thrombus formation at even the highest examined dose (200 mg/kg), while bleeding time was prolonged at even the lowest dose (50 mg/kg). These results suggest that a dosage of antithrombotic agent that does not increase bleeding tendency can be easily established using ticlopidine, although it is relatively difficult using aspirin.
The effects of chondroitin polysulfate (CPS), a semi-synthetic oversulfated chondroitin sulfate, on the metabolism of extracellular matrix in human skin cells were examined. CPS accelerated the production of glycosaminoglycans (GAGs) in human dermal fibroblasts in a dose dependent manner (0.1-3.0 micrograms/ml), but did not modulate the production of noncollagenous protein and collagen, or [3H]-thymidine incorporation into DNA. However, higher concentrations (> 10 micrograms/ml) of CPS suppressed the [3H]-thymidine uptake by fibroblasts. More than 80% of total synthesized GAGs were found to be hyaluronate, whereas CPS did not alter their composition. These results indicated that CPS preferentially enhances the production of GAGs in human dermal fibroblasts, and that GAGs and DNA synthesis in the fibroblasts are independently regulated. Furthermore, CPS might be a unique material able to maintain the level of GAGs in the human skin.
To clarify the relationship between cytokines and arachidonic acid metabolites, we measured tumor necrosis factor (TNF-alpha), interleukin 8 (IL-8), and leukotriene B4 (LTB4). The subjects consisted of 30 patients with sepsis. The results were compared between patients who died (Group A) and those who survived (Group B). All TNF-alpha, IL-8, and LTB4 levels were significantly higher in Group A than in Group B, reflecting the severity of the disease. The LTB4 levels were significantly correlated with the TNF-alpha level and the IL-8 level. These results suggest that inflammatory cytokines, excessively produced due to inflammatory reactions, stimulate as a mediator the release of arachidonic acid, increasing LTB4 production.
In a search for clinically useful indices of liver microsomal function, the correlation between various ratios of serum thyroid hormones, triiodothyronine (T3) and reverse T3 (rT3), and liver microsomal P-450 was investigated in CCl4-treated and untreated rats. Serum and liver samples were obtained before (untreated group, n = 15) and 4 hr after CCl4 administrations (0.2 ml/kg, s.c.). The CCl4-treated group (n = 11) included five randomly chosen untreated rats. In both groups, the ratios of T3/rT3, rT3/T3, and T3/T3+rT3 correlated significantly with liver cytochrome P-450 levels. From these results, it is suggested that the ratios of T3/T3+rT3, T3/rT3, and rT3/T3 can be used as the indices of liver microsomal P-450 levels and that thyroid hormone metabolism is related to liver cytochrome P-450, as was evidenced in untreated animals.
