Alpha-tocopherol modulates lipoprotein cytotoxicity in obstructive nephropathy.

Oxidative stress in unilateral ureteral obstruction (UUO) contributes to the development of glomerular and tubulointerstitial lesions. The present study investigated whether oxidized low-density lipoprotein (oLDL) contributes to the pathogenesis of kidney injury in UUO, and whether alpha-tocopherol modulates such cytotoxicity and promotes repair. Male Sprague-Dawley rats weighing 100-125 g were assigned to three groups of 6 animals each: (1) sham, regular chow; (2) UUO, regular chow; and (3) UUO, alpha-tocopherol supplementation. We found a significant increase in the level of oxidative stress in the UUO group as measured by malondialdehyde (MDA) content in both plasma and kidneys. The LDL isolated from this group was cytotoxic to rat mesangial cells. The level of oxidation and cytotoxicity was significantly reduced when animals were treated with alpha-tocopherol. Plasma cholesterol concentration, kidney MDA, and transforming growth factor beta1 mRNA expression were all significantly increased in the UUO animals, and partially reduced in alpha-tocopherol-treated animals. Our data suggest that oxidative modification of LDL is associated with the renal injury in UUO. Taken together, our data support the concept that alpha-tocopherol can modulate LDL oxidation and its cytotoxic effects on rat mesangial cells in vitro.

Role of brown bears (Ursus arctos) in the flow of marine nitrogen into a terrestrial ecosystem.

We quantified the amount, spatial distribution, and importance of salmon (Oncorhynchus spp.)-derived nitrogen (N) by brown bears (Ursus arctos) on the Kenai Peninsula, Alaska. We tested and confirmed the hypothesis that the stable isotope signature (δ15N) of N in foliage of white spruce (Picea glauca) was inversely proportional to the distance from salmon-spawning streams (r=-0.99 and P<0.05 in two separate watersheds). Locations of radio-collared brown bears, relative to their distance from a stream, were highly correlated with δ15N depletion of foliage across the same gradient (r=-0.98 and -0.96 and P<0.05 in the same two separate watersheds). Mean rates of redistribution of salmon-derived N by adult female brown bears were 37.2±2.9 kg/year per bear (range 23.1-56.3), of which 96% (35.7±2.7 kg/year per bear) was excreted in urine, 3% (1.1±0.1 kg/year per bear) was excreted in feces, and <1% (0.3± 0.1 kg/year per bear) was retained in the body. On an area basis, salmon-N redistribution rates were as high as 5.1±0.7 mg/m2 per year per bear within 500 m of the stream but dropped off greatly with increasing distance. We estimated that 15.5-17.8% of the total N in spruce foliage within 500 m of the stream was derived from salmon. Of that, bears had distributed 83-84%. Thus, brown bears can be an important vector of salmon-derived N into riparian ecosystems, but their effects are highly variable spatially and a function of bear density.

Intracellular trafficking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann-Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation.

Niemann-Pick C disease (NP-C) is a rare inborn error of metabolism with hepatic involvement and neurological sequelae that usually manifest in childhood. Although in vitro studies have shown that the lysosomal distribution of LDL-derived cholesterol is defective in cultured cells of NP-C subjects, no unusual characteristics mark the plasma lipoprotein profiles. We set out to determine whether anomalies exist in vivo in the cellular distribution of newly synthesized, HDL-derived or LDL-derived cholesterol under physiologic conditions in NP-C subjects. Three affected and three normal male subjects were administered [14C]mevalonate as a tracer of newly synthesized cholesterol and [3H]cholesteryl linoleate in either HDL or LDL to trace the distribution of lipoprotein-derived free cholesterol. The rate of appearance of free [14C]- and free [3H]cholesterol in the plasma membrane was detected indirectly by monitoring their appearance in plasma and bile. The plasma disappearance of [3H]cholesteryl linoleate was slightly faster in NP-C subjects regardless of its lipoprotein origin. Appearance of free [14C] cholesterol ill the plasma (and in bile) was essentially identical in normal and affected individuals as was the initial appearance of free [3H]cholesterol derived from HDL, observed before extensive exchange occurred of the [3H]cholesteryl linoleate among lipoproteins. In contrast, the rate of appearance of LDL-derived free [3H]cholesterol in the plasma membrane of NP-C subjects, as detected in plasma and bile, was retarded to a similar extent that LDL cholesterol metabolism was defective in cultured fibroblasts of these affected subjects. These findings show that intracellular distribution of both newly synthesized and HDL-derived cholesterol are essentially unperturbed by the NP-C mutation, and therefore occur by lysosomal-independent paths. In contrast, in NP-C there is defective trafficking of LDL-derived cholesterol to the plasma membrane in vivo as well as in vitro. The in vivo assay of intracellular cholesterol distribution developed herein should prove useful to quickly evaluate therapeutic interventions for NP-C.

Disappearance of two major phosphatidylcholines from plasma is predominantly via LCAT and hepatic lipase.

Metabolism of 1-stearoyl-2-arachidonyl-phosphatidyl-choline (SAPC), a major phosphatidylcholine (PC) species in rat plasma, was compared with 1-palmitoyl-2-linoleoyl-PC (PLPC) metabolism. High-density lipoproteins containing SAPC and PLPC tracers labeled in the sn-2 fatty acid with 3H and 14C isotopes, respectively, were administered. The rats were depleted of endogenous bile acids and infused via the ileum with individual bile acids that ranged widely in hydrophobicity. The half-lives for SAPC and PLPC in plasma were 48 and 57 min, respectively. Most of the 3H activity that disappeared from plasma at 1 h was found in the liver in 1-palmitoyl-2-arachidonyl-PC, SAPC, and 1-oleoyl-2-arachidonyl-PC, indicating phospholipase A1 hydrolysis of plasma SAPC forming 2-arachidonyl-lysophosphatidylcholine, which was reacylated in the liver. Plasma PLPC also underwent phospholipase A1 hydrolysis, as reported previously. The fraction of 3H dose that accumulated in plasma cholesteryl arachidonate was two- to threefold higher than the fraction of 14C dose in cholesteryl linoleate. Multicompartmental models for SAPC and PLPC were developed that included lysophosphatidylcholines and cholesteryl esters. Bile acids did not influence plasma PC metabolism. Lecithin-cholesterol acyltransferase and phospholipase A1 (hepatic lipase) hydrolysis accounted for > or = 90% of the SAPC and PLPC that disappeared from plasma; SAPC and PLPC are comparable as substrates for hepatic lipase, but SAPC is preferred by lecithin-cholesterol acyltransferase.

Antinociceptive effect of purine nucleotides.

The antinociceptive effect of purine nucleotides administered systematically (sc) was determined using the formalin and writhing tests in adult male albino mice. The mechanisms underlying nucleotide-induced antinociception were investigated by preinjecting the animals (sc) with specific antagonists for opioid (naloxone, 1 mg/kg), purinergic P1 (caffeine, 5, 10, of 30 mg/kg); theophylline, 10 mg/kg) or purinergic P2 receptors (suramin, 100 mg/kg; Coomassie blue, 30-300 mg/kg; quinidine, 10 mg/kg). Adenosine, adenosine monophosphate (AMP), diphosphate (ADP) and triphosphate (ATP) caused a reduction in the number of writhes and in the time of licking the formalin-injected paw. Naloxone had no effect on adenosine- or adenine nucleotide-induced antinociception. Caffeine (30 mg/kg) and theophylline (10 mg/kg) reversed the antinociceptive action of adenosine and adenine nucleotide derivatives in both tests. P2 antagonists did not reverse adenine nucleotide-induced antinociception. These results suggest that antinociceptive effect of adenine nucleotides is mediated by adenosine.

Antinociceptive effect of purine nucleotides

The antinociceptive effect of purine nucleotides administered systemically (sc) was determined using the formalin and writhing tests in adult male albino mice. The mechanisms underlying nucleotide-induced antinociception were investigated by preinjecting the animals (sc) with specific antagonists for opioid (naloxone, 1 mg/kg), purinergic P1 (caffeine, 5, 10 or 30 mg/kg); theophylline, 10 mg/kg) or purinergic P2 receptors (suramin, 100 mg/kg; Coomassie blue, 30-300 mg/kg; quinidine, 10 mg/kg). Adenosine, adenosine monophosphate (AMP), diphosphate (ADP) and triphosphate (ATP) caused a reduction in the number of writhes and in the time of licking the formalin-injected paw. Naloxone had no effect on adenosine- or adenine nucleotide-induced antinociception. Caffeine (30 mg/kg) and theophylline (10 mg/kg) reversed the antinociceptive action of adenosine and adenine nucleotide derivatives in both tests. P2 antagonists did not reverse adenine nucleotide-induced antinociception. These results suggest that the antinociceptive effect of adenine nucleotides is mediated by adenosine.

Selective composition of biliary phosphatidylcholines is affected by secretion rate but not by bile acid hydrophobicity.

Little is known about the mechanisms of: 1) biliary phosphatidylcholine (PC) secretion by the hepatocyte, 2) selectivity for biliary 1-palmitoyl-2-linoleoyl-PC (PLPC) secretion, and 3) exclusion of 1-stearoyl-2-arachidonyl-PC (SAPC) from bile. The experiments were designed to determine, in rats, whether selectivity (for PLPC and against SAPC) is influenced by bile acid hydrophobicity or secretion rate. We examined the effects of bile acid depletion and of ileal infusion of taurocholic acid, tauroursodeoxycholic acid, and taurochenodeoxycholic acid. Compared to bile acid depletion, infusion of each bile acid caused PLPC to decrease from 59% of bile PC to 48%, and SAPC to increase from 2.6% to 5%. Bile acid hydrophobicity had no effect on PC selectivity, but selectivity decreased to a moderate degree as total PC secretion increased. To determine whether selectivity is for preformed molecular species, we used a new method to isotopically label four species of hepatic PC. This was done by intravenous injection of PLPC and SAPC labeled in the linoleate (14C) and arachidonate (3H) moieties. Assuming rapid mixing of each PC species in the hepatocyte as supported by the specific activity data, bile SAPC and SLPC were derived entirely from hepatic preformed SAPC and SLPC; bile PLPC was from both preformed PLPC (55%) and an unlabeled input (45%, probably direct secretion of newly synthesized PLPC). In conclusion, the selective composition of bile PC is not related to bile acid hydrophobicity, but is partially lost as secretion increases within the physiologic range.

Gallbladder mucin, arachidonic acid, and bile lipids in patients who develop gallstones during weight reduction.

BACKGROUND: Arachidonic acid (AA) and hydrophobic bile salts (BS) stimulate gallbladder mucin (GBM) secretion, which is thought to be an essential step in gallstone pathogenesis. The present study was performed to evaluate the relationship between AA, BS, and GBM in patients who develop gallstones following weight reduction. METHODS: Eleven patients who underwent gastric bypass, developed symptomatic gallstones, and then underwent cholecystectomy were evaluated. Gallbladder bile was obtained for analysis during each procedure. Matched patients who did not develop gallstones following gastric bypass served as controls. RESULTS: GBM increased in every patient who developed stones (mean increase: 5000%). The largest increase was observed soon after gastric bypass, and this declined curvilinearly with time. Gallbladder bile cholesterol was initially elevated but then rapidly declined before increasing back to pregastric bypass levels after weight loss was complete. No significant changes in phosphatidylcholine molecular species (including AA) or BS composition were observed following weight reduction. Concentrations of cholesterol, phospholipids, and changes in [AA] over time were each a linear function of [BS]. No relationship between GBM and any of these bile constituents was apparent. CONCLUSIONS: These observations strongly suggest that increases in GBM, which occur with gallstone formation in humans, are not the result of alterations in biliary AA or BS composition.

Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.

Our aim was to identify and quantitate cholesterol pools and transport pathways in blood and liver. By studying bile fistula subjects, using several types of isotopic preparations, simultaneous labeling of separate cholesterol pools and sampling all components of blood and bile at frequent intervals, we developed a comprehensive multicompartmental model for cholesterol within the rapidly miscible pool. Data in six components (bile acids, esterified cholesterol in whole plasma, and free cholesterol in blood cells, bile, alpha lipoproteins, and beta lipoproteins) were modeled simultaneously with the SAAM program. The analysis revealed extensive exchange of free cholesterol between HDL and liver, blood cells, and other tissues. There was net free cholesterol transport from HDL to the liver in most subjects. The major organ that removed esterified cholesterol from blood was the liver. A large portion (4,211 mumol) of total hepatic cholesterol comprised a pool that turned over rapidly (t1/2 of 72 min) by exchanging mainly with plasma HDL and was the major source of bile acids and biliary cholesterol. Only 6% of hepatic newly synthesized cholesterol was used directly for bile acid synthesis: the analysis showed that 94% of newly synthesized cholesterol was partitioned into the large hepatic pool (putative plasma membrane free cholesterol) which exchanged rapidly with plasma lipoproteins. Bile acid synthetic rate correlated directly with the size of the large hepatic pool. In conclusion, hepatic and blood cholesterol pools and transports have been quantitated. HDL plays a central role in free cholesterol exchange/transport between all tissues and plasma. In humans, the metabolically active pool comprises a large portion of total hepatic cholesterol that, in part, regulates bile acid synthesis.

Plasma 1-palmitoyl-2-linoleoyl phosphatidylcholine. Evidence for extensive phospholipase A1 hydrolysis and hepatic metabolism of the products.

1-Palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) labeled in either the choline, glycerol, palmitate, or linoleate component in reconstituted rat high density lipoprotein (rHDL), was administered by vein to rats with bile fistula and taurocholate infusion. PLPC disappeared from plasma in a monoexponential fashion with a half-life of 50 min. A small fraction, about 14%, of PLPC disappearance was due to removal of linoleate from the sn-2 ester bond to form plasma cholesterol esters, presumably by lecithin-cholesterol acyltransferase. Otherwise, nearly all of the PLPC components that disappeared from blood in 1 h were recovered in the liver. The choline, glycerol, and linoleate components appeared predominantly in hepatic phosphatidylcholine (PC). These three components remained together in the liver with similar fractions of each in individual PC molecular species, most notably 1-stearoyl-2-linoleoyl-PC and dilinoleoyl-PC as well as PLPC. However, the palmitate component was spread among hepatic triglyceride, free fatty acid, other phospholipids, and all palmitate-containing molecular species of PC. Less than 2% of any administered PLPC component appeared in 1-stearoyl-2-arachidonyl-PC, the major species by mass in the liver. The palmitate component from plasma PLPC appeared in biliary PC at a more rapid rate than glycerol and linoleate components; the latter components appeared in bile in identical fashion. The results show that about two-thirds of plasma PLPC disappearance is due to phospholipase A1 hydrolysis, probably hepatic lipase. The putative produce, 2-linoleoyl-lysoPC, is efficiently reacylated with a saturated fatty acid in the liver, conserving PC.

Effects of R51163 on intake and metabolism in moose.

R51163, a newly synthesized purine alkyl piperidine that produces reliable sedation in cattle, was tested in five adult bull moose (Alces alces). Compared with controls, all animals dosed with 0.4 mg/kg BW ate significantly (P less than 0.05) less dry matter for at least 1 wk after treatment. Median estimates of resting metabolism, measured the day of injection, did not differ between treatment and control groups, although the coefficient of variation was almost two times larger for drugged (15%) versus control (8%) individuals. Dose response was allometric, with larger animals exhibiting longer effects.

Dehydroepiandrosterone and related steroids induce multilamellar lipid structures in cultured human endothelial cells.

Pharmacologic doses of dehydroepiandrosterone (DHEA), a steroid hormone produced naturally by the adrenal cortex, may lower plasma lipoprotein levels in humans and reduce the severity of experimental atherosclerosis in rabbits. Effects of DHEA on cells of the vascular wall, particularly endothelial cells (EC), which are in direct contact with the plasma, have not been documented. The authors have found that micromolar doses of DHEA induce a consistent and reversible morphologic change in cultured EC derived from the human umbilical vein. During 24 hours of exposure to DHEA, cultured EC became loaded with phase-dense, perinuclear cytoplasmic granules, which persisted while DHEA remained in the culture medium. Certain steroids related to DHEA, particularly 17-ketosteroids, also induced perinuclear cytoplasmic granules. The granules lost their phase-density after fixed monolayers were extracted using ethanol or methanol. The granules did not form in media made with lipoprotein-deficient serum, suggesting that serum lipoproteins were involved in formation of the granules. Ultrastructurally, the granules were identical to multilamellar lipid structures, a type of pleomorphic lipid-containing lysosome found in foam cells. The granules were identified as lysosomes by positive reaction for acid phosphatase. The mechanism by which DHEA induces formation of lysosomal lipid structures remains to be determined.

Bile acid production in human subjects: rate of oxidation of [24,25-3H]cholesterol compared to fecal bile acid excretion.

Bile acid production has been quantitated in seven subjects by methods that compare the results of two independent approaches, namely, quantitation of cholesterol side-chain oxidation and fecal bile acid excretion. Six hypertriglyceridemic (HT) subjects and one normolipidemic control were studied by both techniques. A further control subject was studied by the cholesterol side-chain oxidation method alone. Cholesterol side-chain oxidation was quantitated by measuring the appearance of 3H2O after intravenous administration of [24,25-3H]cholesterol, using multicompartmental analysis of plasma cholesterol and [3H]water specific activity. Body water kinetics were independently defined by use of oral D2O. Two HT subjects were restudied while they were taking cholestyramine, 16 g/day. In all ten studies, multicompartmental analysis closely simulated the observed appearance of 3H2O. Values obtained for bile acid production suggest that cholesterol oxidation, or bile acid input, was significantly greater than fecal bile acid output in the HT subjects (P less than 0.05). Cholesterol side-chain oxidation rates in the two normal subjects were lower than those encountered in HT subjects, being similar to published values for normal subjects both for bile acid synthesis as determined by isotope dilution kinetics and fecal bile acid excretion. Studies conducted with two, synthetically different, preparations of [24,25-3H]cholesterol indicated that, in one of the two preparations, approximately 20% of the tritium label was at positions proximal to C24. In the other preparation examined, all of the tritium was located at, or distal to, C24. Further studies revealed that 0.055-0.24% of the dose was present as labile tritium by virtue of its appearance as 3H2O following in vitro incubation with human plasma. Provided these isotope effects are taken into account, multicompartmental analysis of plasma [24,25-3H]cholesterol and body water appears to be a useful technique for quantitating cholesterol oxidation in human subjects.

Baseline body temperatures, heart rates, and respiratory rates of moose in Alaska.

Baseline body temperatures (BT), heart rates (HR) and respiratory rates (RR) were obtained from Alaskan moose (Alces alces gigas Miller) at the Moose Research Center (MRC), Alaska. Excitability, seasons and drugs influenced the values to varying degrees. Excitability was the most influential factor. Safe expected ranges were: BT 38.4 to 38.9 C, HR 70 to 91 beats/min (b/min), and RR 13 to 40 respirations/min (r/min). These ranges incorporated all seasons, a central nervous system depressant drug and a paralyzing drug. Values which may be considered critical and an indication that corrective action should be taken include: BT 40.2 C, HR 102 b/min, and RR 40 r/min. It is recommended that persons trained in monitoring vital signs be on hand during moose capture and immobilization procedures.

Isolation of chlordecone binding proteins from pig liver cytosol.

Cytosolic proteins may play an important role in the transport of water insoluble substances through the cytosol to various subcellular locations. The binding of chlordecone (CD) to pig liver cytosolic proteins was studied after the simultaneous administration of [14C]CD and [3H]cholesterol via the portal vein. The isolation of chlordecone binding proteins (CDBPs), from liver cytosol consisted of repeated ultracentrifugation, ammonium sulfate fractionation, and chromatography on Bio-Gel A 0.5m, carboxymethyl cellulose (CMC), and Sephadex G-100. Three proteins retained [14C]CD after elution from the CMC column. CDBP I and CDBP II also retained [3H]cholesterol through this stage of purification, while CDBP III did not. The molecular weights, estimated by Sephadex G-100 gel filtration, were 33,500 and 67,000 for CDBP IA and CDBP IB, 49,000 for CDBP II, and 44,000 for CDBP III. Since dissociation of bound cholesterol could not be avoided during the purification procedures, binding of cholesterol to the CDBPs eluted from Sephadex G-100 was investigated. Incubation of CDBPs with [3H]cholesterol resulted in a 2000-fold increase of 3H associated with CDBP II, an 800-fold increase with CDBP IA, a 100-fold increase for CDBP IB, and a 300-fold increase for CDBP III. The isolation characteristics, molecular weights, and cholesterol binding properties of the CDBPs are compared with cytosolic cholesterol binding proteins previously isolated. The high specific activity binding of both CD and cholesterol by CDBP I and CDBP II suggests that CD and cholesterol share a common transport pathway in the liver cytosol.

High-density lipoproteins decrease the biliary concentration of chlordecone in isolated perfused pig liver.

Chlordecone (CD) is an organochlorine pesticide associated with albumin and high-density lipoproteins (HDL) in the plasma. It is found in higher concentrations in the liver than in other tissues and is excreted in the bile. The influence of plasma HDL on the biliary excretion of CD was studied using isolated pig liver perfused with a Krebs-Ringer bicarbonate solution containing albumin, dextrose, and pig red blood cells. Within 5 min after administration into the perfusion medium of [14C]CD bound to albumin or to HDL, only 13% of the [14C]CD dose remained in the perfusate, showing that CD is rapidly taken up by the liver. After 60 min the plasma concentration was constant at 0.008% dose/ml when [14C]CD was administered bound to albumin in the absence of HDL and at 0.004% dose/ml when administered bound to HDL. The mean concentration of CD in the bile was higher when CD was administered bound to albumin in the absence of HDL (0.039% dose/ml) than when it was administered bound to HDL (0.010% dose/ml). The elimination rate constant of CD from the liver into the bile was 0.007/min whether CD was administered bound to albumin or to HDL. The addition of HDL to the perfusion system after the administration of albumin-bound CD resulted in lower biliary CD concentrations. The results suggest that HDL affects the distribution of CD between the perfusate and liver and between liver and bile. In both cases, distribution toward the liver is favored.

Blue-green color and composition of Stejneger's beaked whale (Mesoplodon stejnegeri) milk.

Two hundred ml of milk were obtained from a lactating Stejneger's beaked whale stranded at Ninilchik, Alaska on 21 Oct, 1980. Total solids (41%) were similar to values reported for sperm and belukha whales, while fat (17%) was half as great and crude protein (17%) was 2-4 times greater than in milk of these species. Lactose was not detected. Calcium (0.22%) was greater than reported for pigmy sperm whales but less than for blue whales. Phosphorus (0.07%) was less than for any of the above species. Sodium and potassium concentrations were 0.13% and 0.11%, respectively. Values (microgram/g) for other elements analyzed (magnesium, 42; iron, 35; copper, 2.6; zinc, 1.5; manganese, 0.3; selenium, 0.36) have not been reported for whale milk. Based on SDS-gel electropherograms, this whale milk did not contain a whey protein corresponding to cattle milk alpha-lactalbumin. A blue-green pigment in the milk was identified as biliverdin.