Interferon crosses blood-cerebrospinal fluid barrier in monkeys.

Interferon was detected in the cerebrospinal fluid (CSF) of monkeys injected iv or im with 30 million units of human leukocyte interferon. The im injection maintained a long-lasting plateau at about 1/30th of the corresponding level of interferon in the serum. Interferon injected into the serum. Interferon injected into the cerebrospinal canal was cleared from CSF at a similar rate as it disappeared from blood after iv administration of a high dose. A relatively stable serum level was maintained for 12-24 hr after the injection of interferon into the CSF space.     

Tissue distribution and plasma clearance of heparin-binding EGF-like growth factor (HB-EGF) in adult and newborn rats

Heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells from various forms of injury in vitro and attenuate intestinal ischemia/reperfusion damage in vivo. With the goal of eventual clinical use of HB-EGF to protect the intestines from injury in neonates, children, and adults, the pharmacokinetics and biodistribution of 125I-labeled HB-EGF were investigated. After intravenous bolus, HB-EGF had a distribution half-life of 0.8 min and an elimination half-life of 26.67 min. After gastric administration, the bioavailability was 7.8%, with a 2.38 h half-life in the absorption phase and an 11.13 h half-life in the elimination phase. After intravenous dosing, most radioactivity was found in the plasma, liver, kidneys, bile, and urine, whereas it was mainly distributed in the gastrointestinal tract after intragastric administration. The degradation of 125I-HB-EGF in plasma from newborn rats was lower than that in adult rats after gastric administration. This supports the feasibility of enteral administration of HB-EGF in the treatment of gastrointestinal diseases, including newborns afflicted with necrotizing enterocolitis.     

Clearance of chicken cystatin from the rat circulation

Chicken cystatin (mixed form) was prepared from egg white. Radioactively labeled preparations were administered intravenously to rats. ¹²⁵I-cystatin disappeared from the rat circulation with a half-life of -73 min. The radiolabeled inhibitor was rapidly taken up by the kidneys. Percoll density gradient analysis showed that it was incorporated into lysosomes. Within 24 hr after the injection of ¹²⁵I-cystatin, 25% of the administered radioactivity was recovered in the urine, but only 2% was in the protein-bound form.     

Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat

The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of 125I-rhIL-6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half-life of 3 min, and of a second slow one corresponding to a half-life of about 55 min. By cellulose-acetate electrophoresis it was shown that rhIL-6 binds to a plasma protein resulting in a complex migrating in the beta-gamma region; 20 min after intravenous injection, about 80% of the 125I-rhIL-6 that had disappeared from the circulation was found in the liver. 125I-rhIL-6 was exclusively localized on the surface of parenchymal cells suggesting the existence of an interleukin-6 receptor on the hepatocytes.     

Clearance of circulating desialylated thyroglobulins in the rat.

Canine and rat thyroglobulins were labeled with 125I either in vitro or in vivo, and were utilized for plasma clearance studies performed with rat. The half-life of physiologically radioiodinated asialothyroglobulins was about 6 min, while that of chemically radioiodinated asialothyroglobulin was about 12 min. No marked species difference was observed in this clearance. The label which had disappeared from the blood was recovered mainly in the liver, and this uptake was blocked by the simultaneous injection of desialylated orosomucoid but not by native orosomucoid. Radiolabeled monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine were detected in the liver 17 min after intravenous injection of asialothyroglobulin labeled with 125I in vivo, suggesting the possible production of thyroid hormones in extrathyroidal tissues.     

Dopamine β-hydroxylase: Determination of half-life and appearance in lymph fluid after IV injection of 125I-DBH into rats

A 4 day half-life of dopamine beta-hydroxylase (DBH) was determined for rats injected IV with ¹²⁵I-rat DBH from the slow exponential component of radioactivity appearing in plasma, urine, feces and combined urine and feces. Half-life estimates for ¹²⁵I-rat DBH injected IV into WKY and SHR animals did not differ from Sprague Dawley (Zivic Miller) rats. Radioactivity declined in parallel in plasma, urine and feces following IV ¹²⁵I-rat DBH administration and each radioactivity falloff curve could be resolved into two components. The slow phase of the decline of radioactivity excreted into urine and feces from which DBH half-life was calculated occurred between 5 and 25 days after ¹²⁵I-rat DBH injection. The early fast phase which is associated with distribution of the exogenous protein in body fluids and tissues continued for approximately the first 140 hr after DBH injection. The distribution characteristics of IV administered active bovine DBH and ¹²⁵I-rat DBH into the lymphatic system were examined. After active bovine DBH or ¹²⁵I-rat DBH was injected IV into rats, active DBH or radioactivity, respectively, appeared in lymph fluid (thoracic duct) within 20 min; reached peak concentrations within 90 min, and thereafter, declined in parallel with the plasma concentration. The concentration of radioactivity in plasma and lymph fluid were found to be unequal at 9 hr but were equivalent 68–75 hrs after IV injection of ¹²⁵I-rat DBH. Based on the amount of active DBH or radioactivity which accumulates in lymph fluid it is clear that'a substantial amount (> 50%) of the DBH in blood circulates through the lymphatic channels. Analysis of parallel experiments with labelled serum albumin indicate that use of these methods to study plasma proteins do provide sensitive measures of biological half-life and lymphatic distribution characteristics. Specifically for DBH, the results of our study suggest that DBH normally circulates in plasma and lymph fluid with a biological half-life of 4 days.     

Time course of distribution to tissues of 125I-somatomedin A injected into the rat

125I-somatomedin A (SMA) was injected iv into rats. Distribution studies in rats showed concentrations of radioactivity to be high in kidney and plasma, low in brain, and intermediate in other tissues. The concentration of total and trichloracetic acid (TCA) precipitable radioactivity in rat blood and tissues fell at rapid rate. Ninety per cent of the radioactivity was in the urine in 24 hr, and only 15% of urine radioactivity was TCA precipitable. The half-life of the radioactivity in TCA-precipitable fraction from blood and that from tissues were nearly identical (about 6 hr). In both liver and kidney, TCA-precipitable radioactivity was detected in membrane and/or organellar fraction and cytosol fraction. Sephadex G-200 chromatography at neutral PHY AT NEUTRAL PH of plasma after injection of 125I-SMA revealed 3 peaks of radioactivity in higher molecular weight region than purified SMA.     

Clearance of circulating desialylated thyroglobulins in the rat

Canine and rat thyroglobulins were labeled with ¹²⁵I either in vitro or in vivo, and were utilized for plasma clearance studies performed with rat. The half-life of physiologically radioiodinated asialothyroglobulins was about 6 min, while that of chemically radioiodinated asialothyroglobulin was about 12 min. No marked species difference was observed in this clearance. The label which had disappeared from the blood was recovered mainly in the liver, and this uptake was blocked by the simultaneous injection of desialylated orosomucoid but not by native orosomucoid. Radiolabeled monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine were detected in the liver 17 min after intravenous injection of asialothyroglobulin labeled with ¹²⁵I in vivo, suggesting the possible production of thyroid hormones in extrathyroidal tissues.     

Spin trapping agent, phenyl N-tert-butylnitrone, reduces nitric oxide production in the rat brain during experimental meningitis

Phenyl N-tert-butylnitrone (PBN) is a spin trapping agent previously shown to exert a neuroprotective effect in infant rat brain during bacterial meningitis. In the present study, we investigated the effect of systemic PBN administration on nitric oxide (NO) production in a rat model of experimental meningitis induced by lipopolysaccharide (LPS). We assessed the NO concentration in rat brain tissues with an electron paramagnetic resonance (EPR) NO trapping technique. In this model, rats receiving intracisternal LPS administration showed symptoms of meningitis and cerebrospinal fluid (CSF) pleocytosis. The time course study indicated that the concentration of NO in the brain reached the maximum level 8.5h after injection of LPS, and returned to the control level 24 h after the injection. When various doses of PBN (125-400 mg/kg) were injected intraperitoneally 30 min prior to LPS, NO production in the brain was reduced with increasing PBN dose (250 mg/kg suppressed 80% at 8.5h after LPS injection), and white blood cells (WBC) in CSF were significantly decreased. We concluded that reduction of NO generation during bacterial meningitis contributes to the neuroprotective effect of PBN in addition to its possible direct scavenging of reactive oxygen intermediate (ROI).     

Biochemical and morphological modifications in rabbit Achilles tendon during maturation and ageing.

1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow.     

Glucagon kinetics in growing rats fed different levels of protein and/or energy

The present work was carried out to evaluate the kinetic parameters of glucagon in growing rats divided into three groups: T, H and E. Group T (Control group) was fed a control diet (crude protein: 11.8%). Groups H and E received a high protein diet (crude protein: 19%) distributed in either equal (Group H) or restricted amounts (Group E) with respect to the control. Thus, the main characteristic of Group H was the high level of protein intake (+ 68%) when Group E rats underwent a moderate increase in protein intake but a striking caloric deprivation (-25%). In all cases, the animals were fed a meal every 4 hours. The kinetic parameters of glucagon metabolism were estimated from the plasma disappearance curves of 125I-glucagon for five minutes following a pulse injection of purified 125I-glucagon (1 muCi, about 3.8 ng/100 g BW). Plasma 125I-glucagon was measured after gel filtration of plasma on Biogel P-10. Tissue radioactivity (mainly liver and kidneys) was recorded seven minutes after 125I-glucagon injection. The results showed that the plasma 125I-glucagon level was higher in Group H than in the other groups 1 min after the injection. At all other times (2, 3.5 and 5 min) it was similar in all groups. 125I-glucagon was rapidly cleared from plasma and rapidly taken up by the liver and kidneys. In the 3 experimental groups, mean half-life and metabolic clearance rate were estimated to be 2 min and 6 ml/min/100 g BW, respectively. Excess protein intake resulted in a reduction in the apparent initial distribution volume of 125I-glucagon without modifying significantly its turn-over rate and metabolic clearance rate. Kidneys and liver (6% BW) accounted for about 20% of the 125I-glucagon uptake by tissues 7 min after injection. Group H kidneys and liver were more labelled than in other groups. These results suggest that increased protein intake (without further caloric deprivation) can induce some changes in glucagon metabolism which could partially contribute to the increase in glucagonemia usually observed in animals fed high protein diets.     

The presence of cocaine and benzoylecgonine in rat cerebrospinal fluid after the intravenous administration of cocaine.

Cocaine hydrochloride, in doses of 0.5, 1.0, 2.0 and 4.0 mg/kg, iv, was administered to male Sprague-Dawley rats. Cerebrospinal fluid (CSF) was collected from the cisterna magna over a 20 min period and blood samples were obtained at 20 min after cocaine administration. In addition, blood samples for the 1 mg/kg dose of cocaine were collected at 2, 10, 20 and 30 min following drug injection. Gas chromatography/mass spectrometry was used for the analysis of cocaine and its metabolites in plasma and CSF. The disappearance of cocaine (1 mg/kg) from the plasma exhibited first order kinetics with a half-life of 18.11 +/- 3.22 min. Cocaine and benzoylecgonine were found in CSF and the concentrations of cocaine and benzoylecgonine increased in CSF as the doses of cocaine were increased. CSF flow rates were not altered by the iv administration of cocaine or benzoylecgonine. The CSF-to-plasma ratios for cocaine were quite similar to each other over the dosage range of cocaine that was administered; however, the CSF-to-plasma ratios for benzoylecgonine decreased as the concentrations of benzoylecgonine increased in plasma and CSF. When benzoylecgonine (2 mg/kg, iv) was given, the compound was detected in CSF indicating that benzoylecgonine can enter into the central nervous system from the peripheral blood. This investigation shows that cocaine and benzoylecgonine can be assayed in CSF and that the plasma levels of these compounds correlate with their concentrations in CSF.     

Lack of recognition of Nepsilon-(carboxymethyl)lysine by the mouse liver reticulo-endothelial system: implications for pathophysiology

Advanced glycation end products (AGEs) are known to be associated with a number of pathological conditions, such as diabetes mellitus, Alzheimer's disease, uremia, as well as with normal aging. This study was undertaken to investigate whether Nepsilon-(carboxymethyl)lysine (CML), a major structure among numerous AGEs, engenders hepatic AGE clearance. For this purpose uptake of BSA substituted with heterogeneous AGEs or with CML only was monitored in vivo and in cultured hepatic scavenger cells. Here, we show that following intravenous administration of 125I-AGE-BSA and 125I-CML-BSA, blood radioactivity was reduced by 50% after 50s and >100 min, respectively. Recoveries from the circulation at 6 min after injection were: 5% for AGE-BSA, 95% for CML-BSA. More than 80% of the injected AGE-BSA was recovered from the liver. AGE-BSA, but not CML-BSA, was avidly endocytosed by cultured liver scavenger cells. Our results suggest that CML does not engender AGE-BSA clearance. Macromolecules substituted with CML only may escape elimination and cause pathological effects.     

The effects of different anaesthetic treatments on the adreno-cortical functions and glucose levels in NZW rabbits

The effects of five anaesthetics on the corticosterone, cortisol and glucose concentrations were investigated in the NZW rabbit. Sixty animals were assigned to 6 treatment groups (n= 10 per group): control ( iv saline solution injection), ketamine (10 mg/kg iv) with either xylazine (3 mg/kg iv) or diazepam (2 mg/kg iv), pentobarbitone (30 mg/kg iv), thiopentone (20 mg/kg iv) and fentanyl/droperidol (1 mg/kg sc). Plasma glucocorticoids were measured by competitive enzymeimmunoassay EIA and glucose by an autoanalyzer, previously validated for this species in both cases. Blood samples were obtained at 6 time-points: before injection, at 10, 30, 60, 120 min and 24 h after injection of the anaesthetics/saline. A significant decrease of plasma glucocorticoids at 10-60 min was observed in the pentobarbitone and fentanyl/ droperidol groups, whereas the administration of ketamine/diazepam or thiopentone stimulated plasma glucocorticoid release, principally in the recovery period. However, in the ketamine/xylazine group no changes were observed in the glucocorticoid levels, except for a significative increase of cortisol at 60-120 min. Glucose levels significantly increased after ketamine/diazepam administration and principally, after ketamine/xylazine treatment. The present data suggest that ketamine/xylazine has little effect on glucocorticoid levels and provides an adequate level of surgical anaesthesia, hence it would be the anaesthetic of choice, although the hyperglycaemic effect after injection has to be considered for any experimental procedures in rabbits.     

Ghrelin is suppressed by glucagon and does not mediate glucagon-related growth hormone release

BACKGROUND: Glucagon stimulation is routinely used as a provocative test to assess growth hormone (GH) sufficiency in pediatrics. Ghrelin also markedly stimulates GH secretion. Because glucagon stimulates the promoter of the ghrelin gene in vitro as well as ghrelin secretion by the perfused rat stomach, we sought to determine whether ghrelin mediates glucagon-induced GH secretion. METHODS: We compared ghrelin, GH, insulin and glucose responses following administration of 0.03 mg/kg intravenously (iv; max. 1 mg) and 0.1 mg/kg intramuscularly (im; max. 2 mg) of glucagon in two groups (n = 10-11/group) of GH-sufficient children. We also measured ghrelin before and 6 min after iv administration of 1 mg glucagon in 21 adult subjects. RESULTS: In children, glucagon caused a 26% decrease in ghrelin and a 72% increase in glucose concentrations that were independent of the dose or administration route of glucagon. In contrast, the insulin response was 2-3 times higher following administration of 0.1 mg/kg im compared to 0.03 mg/kg of glucagon iv. There was a significant correlation between the maximum decrease in ghrelin and increases in glucose (p = 0.03) but not in insulin. There was a significant correlation between ghrelin and GH area under the curve after controlling for the dose of glucagon (p = 0.03) but not for the maximum increase in glucose.In normal adults, glucagon administration caused a 7% decrease in ghrelin concentrations after 6 min (p = 0.0002). CONCLUSION: Ghrelin does not play a causal role in the GH response to pharmacological glucagon administration, which suppresses ghrelin levels starting a few minutes after injection.     

Spin trapping agent,phenyl N-tert-butylnitrone,reduces nitric oxide production in the rat brain during experimental meningitis

Phenyl N-tert-butylnitrone (PBN) is a spin trapping agent previously shown to exert a neuroprotective effect in infant rat brain during bacterial meningitis. In the present study, we investigated the effect of systemic PBN administration on nitric oxide (NO) production in a rat model of experimental meningitis induced by lipopolysaccharide (LPS). We assessed the NO concentration in rat brain tissues with an electron paramagnetic resonance (EPR) NO trapping technique. In this model, rats receiving intracisternal LPS administration showed symptoms of meningitis and cerebrospinal fluid (CSF) pleocytosis. The time course study indicated that the concentration of NO in the brain reached the maximum level 8.5h after injection of LPS, and returned to the control level 24 h after the injection. When various doses of PBN (125–400 mg/kg) were injected intraperitoneally 30 min prior to LPS, NO production in the brain was reduced with increasing PBN dose (250 mg/kg suppressed 80% at 8.5h after LPS injection), and white blood cells (WBC) in CSF were significantly decreased. We concluded that reduction of NO generation during bacterial meningitis contributes to the neuroprotective effect of PBN in addition to its possible direct scavenging of reactive oxygen intermediate (ROI).     

The effects of insulin and glucose on the induction and intracellular translocation of delta-aminolevulinic acid synthase

The administration of insulin and glucose to young Sprague-Dawley rats (125-150 g) resulted in changes in the intracellular distribution and in the turnover rates of delta-aminolevulinic acid synthase (ALAS) activity in the mitochondria and the cytosol. When starved, allylisopropylacetamide (AIA)-induced rats were injected with either insulin or glucose, the percentage of the total ALAS activity found in the cytosol increased from 27% in control animals to 33-40% in insulin-treated and 50% in glucose-treated rats. Similar increases of the ALAS activity in the cytosol were observed after insulin treatment of noninduced, starved animals. Glucose administration also repressed 25-40% of the AIA induction of ALAS as previously reported; however, this effect apparently was not a result of elevated insulin levels, since there was no observed repression of AIA induction after insulin administration. The effects of insulin and glucose on the turnover rates of ALAS activity in the mitochondria and in the cytosol were investigated by observing changes in the half-lives of ALAS activity in the two intracellular compartments. Administration of both insulin and glucose resulted in an increased half-life of ALAS activity in the cytosol from 20.8 to over 100 min, while the mitochondrial half-life was not significantly changed. When insulin was given to either fed, AIA-induced or to starved, noninduced rats, the half-life of the cytosolic ALAS increased from about 14 to 40 min. In contrast to the starved, induced animals, the mitochondrial ALAS half-life in starved, noninduced animals decreased 50%. These results suggest that insulin and glucose treatment may inhibit the translocation of ALAS from the cytosol into the mitochondrial matrix.     

Studies on the effect of hepatectomy on pig post-heparin plasma lipases

The effect of different amounts of heparin injected intravenously in swine on lipoprotein lipase and hepatic lipase activities in post-heparin plasma was studied using an immunochemical method. After the injection of 50 I.U. of heparin/kg body weight the apparent half-life of lipoprotein lipase and hepatic lipase activity measurable in post-heparin plasma was 15 min. This was prolonged to more than 60 min after the injection of 1000 I.U./kg body weight. It is concluded that the higher the heparin dose injected the longer can lipolytic activities be measured in plasma. A possible explanation for these findings is that the amount of circulating heparin governs the distribution of lipoprotein lipase and hepatic lipase between an endothelial-bound form and a circulating form and thus determines the apparent ‘half-life’ of lipase activity measurable in plasma. The apparent half-life of radioactively labelled heparin in normal swine was not different from that observed in hepatectomized animals. After hepatectomy no immunoreactive hepatic lipase activity could be demonstrated in post-heparin plasma confirming our previous findings that the liver is the only source of hepatic lipase.To study the role of the liver in the clearance of plasma lipoprotein lipase activity after the administration of heparin normal and hepatectomized pigs were given 200 I.U./kg body weight followed by a heparin infusion of 100 I.U./ h per kg body weight. In the control pigs the heparin injection caused a rapid release of lipoprotein lipase and hepatic lipase activities. These activities were maintained in the circulation during the 3-h infusion at a level of about 60% of the levels measurable 30 min after the injection. In hepatectomized pigs the lipoprotein lipase activity rose during the infusion to about six times the activity recorded 30 min after heparin administration. From these experiments we conclude that after heparin injection the liver is involved in the clearance of post-heparin plasma lipolytic activity.     

Pretreatment with glucose increases entry of urocortin into mouse brain

Although urocortin is a potent inhibitor of food ingestion after peripheral administration, it was recently shown that under normal conditions this peptide crosses the blood-brain barrier (BBB) at a very slow rate. We examined whether hyperglycemia could stimulate the rate of entry (K(i)) of (125)I-urocortin into the mouse brain. In euglycemic mice, (125)I-urocortin injected iv entered the brain at a rate similar to that of the vascular marker (99m)Tc-albumin. However, injection of glucose (3 g/kg, ip) 0.5, 1, or 2 h before the (125)I-urocortin greatly increased the influx of urocortin. Without the glucose, the self-inhibition characteristic of a saturable transport system was not apparent. Self-inhibition could be demonstrated after the glucose injection, indicating activation of a transport system for urocortin that was saturable. Injection of insulin (10 U/kg, ip) 1 or 2 h before the (125)I-urocortin decreased the K(i). Thus, the entry of urocortin into brain can be activated by changes in the concentration of blood glucose, illustrating the responsiveness of the BBB to regulatory influences.     

Role of nitric oxide in the cerebrovascular and thermoregulatory response to interleukin-1 beta

Central administration of interleukin-1 beta (IL-1 beta) increases cerebral blood flow (CBF) and body temperature, in part, through the production of prostaglandins. In previous studies, the temporal relationship between these effects of IL-1 beta have not been measured. In this study, we hypothesized that the increase in CBF occurs before any change in brain or body temperature and that the cerebrovascular and thermoregulatory effects of IL-1 beta would be attenuated by inhibiting the production of nitric oxide (NO). Adult male rats received 100 ng intracerebroventricular (icv) injection of IL-1 beta, and cortical CBF (cCBF) was measured by laser-Doppler in the contralateral cerebral cortex. A central injection of IL-1 beta caused a rapid increase in cCBF to 133 +/- 12% of baseline within 15 min and to an average of 137 +/- 12% for the remainder of the 3-h experiment. Brain and rectal temperature increased by 0.4 +/- 0.2 and 0.5 +/- 0.2 degrees C, but not until 45 min after IL-1 beta administration. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg iv) completely prevented the changes in cCBF and brain and rectal temperature induced by IL-1 beta. L-Arginine (150 mg/kg iv) partially reversed the effects of L-NAME and resulted in increases in both cCBF and temperature. These findings suggest that the vasodilatory effects of IL-1 beta in the cerebral vasculature are independent of temperature and that NO plays a major role in both the cerebrovascular and thermoregulatory effects of centrally administered IL-1 beta.