Effect of alcohol on urinary and blood dolichols.

Alcohol appears to affect dolichol metabolism, as both serum and urinary dolichol concentrations were found to be significantly higher in alcoholics than in social drinkers. Furthermore, acute heavy drinking (5.5 g alcohol/kg body weight during 42 h) increased urinary dolichol excretion significantly, whereas moderate drinking (60 g/day for 10 days) had no effect. Increased urinary dolichol concentrations in alcoholics returned rapidly to normal with a half-life decay of 3 days, whereas increased serum dolichol concentrations did not change during a 7-day observation period. The mechanism behind alcohol-induced alterations in dolichol metabolism remains unclear, but based on our results, it seems likely that serum and urinary dolichols are regulated independently from each other.     

Chronopharmacological study of furosemide in rats

The present experiment was undertaken to determine whether or not the effects of furosemide depend upon the administration time and, if so, to study the mechanism(s) for these variations. After administration of furosemide (5 mg/kg) in Wistar rats at 10:00 or at 22:00, urine volume and urinary excretion of sodium, furosemide, and prostaglandin E2 (PGE2) were measured. Urine volume and urinary excretion of sodium and furosemide, but not PGE2, were significantly greater when furosemide was administered at 10:00 than when it was administered at 22:00. There was a good correlation between the urinary output of furosemide and the urine volume, or the urinary sodium. It is concluded that the effects of furosemide vary with the administration time and these variations depend upon the amount of furosemide secreted in urine.     

Post-collection, pre-measurement variables affecting VEGF levels in urine biospecimens

Angiogenesis, the development and recruitment of new blood vessels, plays an important role in tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is an important stimulator of angiogenesis. Circulating and urinary VEGF levels have been suggested as clinically useful predictors of tumour behaviour, and investigations into these associations are ongoing. Despite recent interest in measuring VEGF levels in patients, little is known about the factors that influence VEGF levels in biospecimens. To begin to address this question, urine samples were collected from patients with solid tumours undergoing radiotherapy and healthy volunteers. Four factors were examined for their effects on VEGF concentrations as measured by chemiluminescent immunoassay: time from sample collection to freezing, number of specimen freeze-thaw cycles, specimen storage tube type and the inclusion or exclusion of urinary sediment. The results of this study indicate that time to freeze up to 4 hrs, number of freeze-thaw cycles between one and five, and different types of polypropylene tubes did not have statistically significant effects on measured urinary VEGF levels. Urinary sediment had higher VEGF levels than supernatant in five of six samples from healthy patients.It is not clear whether there is an active agent in the sediment causing this increase or if the sediment particles themselves are affecting the accuracy of the assay.Therefore, we recommend centrifuging urine, isolating the supernatant, and freezing the sample in polypropylene microcentrifuge tubes or cryogenic vials within 4 hrs of collection.In addition, we recommend the use of samples within five freeze-thaw cycles.     

Uptake of dolichol by Vero cells.

Characterization and kinetics of dolichol uptake by a Vero cell line are reported. Vero cells incorporate dolichol in a time- and dose-dependent manner. Optimal uptake is found at 37 degrees C and at a pH of 7.4. In contrast to cholesterol, an inhibitory effect on the dolichol incorporation is found for farnesol, geraniol, and retinol. Long chain polyprenols were slightly stimulatory. The translocation seems not to be highly energy dependent. The lack of substantial inhibition by chloroquine does not plead for a receptor-mediated endocytosis. Incorporated dolichol was distributed over both membranes and supernatant fractions, paralleling the distribution of the lysosomal marker beta-N-acetylhexosaminidase. The incorporated dolichol is subject to a fast efflux process, which is potentiated by the presence of lipid acceptors in the extracellular medium.     

Urinary benzene determination by SPME/GC-MS. A study of variables by fractional factorial design and response surface methodology

The urinary excretion of the unmetabolized benzene seems to be a very good index for biomonitoring benzene in occupationally exposed people. The use of solid phase microextraction (SPME) offers important advantages for its determination. Several variables can influence the benzene extraction process. Experimental design methodology was used to estimate the influence of the different variables and to evaluate the simultaneous effect of the more significant variables on the benzene extraction. The results showed that sample temperature, sample volume and their interaction were the more significant factors. A model was found that relates the amount of benzene extracted with the studied variables. The more adequate working conditions were: extraction temperature 15 degrees C, incubation time 1 min, extraction time 1 min and 2.5 ml of sample volume. The results indicate that this method is capable of providing sensitive and accurate results for the biomonitoring of benzene in urine.     

Alkaline phosphatase isoenzymes in human kidney and urine,immunohistochemical, immunological and biochemical characterization

Summary Human kidney contains two antigenetically distinct isoenzymes of alkaline phosphatase (AP): a liver type and an intestinal type. The intestinal type AP is a minor component (1%–4%) of the total AP activity: it is found only in the cytoplasm. Both isoenzymes are located, found by an immunohistochemical technique, in the proximal convoluted tubules. This histochemical result eliminates the possibility that the low intestinal AP content in the kidney might only originate from blood vessels, where the intestinal isoenzyme was also found. The renal isoenzymes contribute to urinary AP. Intestinal type AP in urine of healthy persons, 10%–40% of the total AP activity, was found after high speed centrifugation predominantly in the supernatant (100,000 g), the liver type mainly in the sediment. Biochemical characterization revealed that intestinal type AP in kidney and urine are identical and differ from the isoenzyme of intestinal mucosa only slightly in their electrophoretic mobility.     

Distribution, metabolism and excretion of [1-14C]dolichol injected intravenously into rats.

[1-14C]Dolichol mixed in vitro with rat serum and injected intravenously into rats was rapidly cleared from the circulation in a manner consistent with a two-compartment model. About 80% of the radioactivity recovered from animals killed after 1 day was in the liver, with smaller amounts being found in lung, carcass (internal organs removed), gastrointestinal tract and contents, and spleen. The kidneys, testes and heart contained little radioactivity, and the brain did not appear to take up any [1-14C]dolichol. The half-life for the turnover of radioactivity from [1-14C]dolichol in tissues varied considerably, being 2 days for the lung, 17 for liver and about 50 days for the carcass. After 1 day, and also after 4 and 21 days, most of the radioactivity in all tissues was as [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester, although a small amount of incorporation of [1-14C]dolichol radioactivity into phospholipids was also observed. Faeces collected over the first 4 days after injection contained 13% of the [1-14C]dolichol dose, but urine and expired air contained only small amounts of radioactivity. Radioactivity in faeces was nearly all as unchanged [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester. The [1-14C]dolichol remaining in liver after 21 days appeared to be in a pool (possibly lysosomes) where most of it was not subject to excretion.     

Separation of Brain Dolichol Kinase from Endogenous Activating Factors: Evidence That Phospholipid Enhances the Interaction Between Enzyme and Dolichol

Porcine brain dolichol kinase activity is effectively solubilized by extracting salt-washed microsomes with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). When the detergent-solubilized activity is chromatographed on Sepharose CL-6B, a low amount of dolichol kinase activity is recovered in the void volume, and a dolichol kinase activator (DKA) is eluted (Ve/Vo = 1.9-2.2) with the bulk of the membrane phospholipids. Although only approximately 20% of the activity applied to the Sepharose CL-6B column is detected in the column fractions, virtually all of the original activity is restored when the Vo fraction is recombined with DKA. Endogenous DKA, isolated from brain microsomes, is heat-stable, is extractable with CHCl3/CH3OH (2:1), and has the chemical and chromatographic properties of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, approximately 50% of the stimulatory activity is lost when the PC present in the DKA fraction is degraded by purified phospholipase C from Clostridium perfringens. Also consistent with a phospholipid co-factor requirement, the dolichol kinase activity recovered in the partially phospholipid-depleted fraction (Vo) is markedly stimulated by various molecular species of exogenous purified PC or PE, but not by phosphatidylinositol, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, or sphingomyelin. A comparison of defined molecular species shows that PCs containing oleoyl or linoleoyl groups in the 1 and 2 positions are the most stimulatory, suggesting that the fatty acyl moieties are involved in the enzyme-phospholipid interaction. Kinetic analyses indicate that PC enhances the interaction between dolichol kinase and dolichol, the lipophilic substrate, but does not alter the apparent Km for CTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The urinary excretion of epidermal growth factor in the rat is reduced by aprotinin, a proteinase inhibitor.

The present study on the rat shows that i.v. administration of the proteinase inhibitor aprotinin reduces the urinary output of immunoreactive epidermal growth factor (EGF) while the amount of immunoreactive EGF in the kidneys is increased. This indicates that the EGF-precursor in the rat kidney in vivo is processed by an aprotinin inhibitable proteinase. EGF is produced in the kidneys as a precursor with a molecular weight of approximately 130 kDa. In rat urine, nanomolar amounts of 6 kDa EGF are excreted per 24 h together with small amounts of high molecular weight forms of EGF. During i.v. administration of aprotinin the median urinary output of immunoreactive EGF is reduced to 15% of the excretion of control rats (23 pmol/2 h versus 157 pmol/2 h, P less than 0.001). Especially the excretion of 6 kDa EGF is reduced (median excretion 12 pmol/2 h versus 134 pmol/2 h, P less than 0.001). The amount of immunoreactive EGF in the kidney tissue is increased after aprotinin administration (median amount 0.11 pmol EGF/mg protein versus less than 0.04 pmol EGF/mg protein, P less than 0.001). Neither the creatinine clearance, the total urinary protein output, nor the volume of urine produced was affected by aprotinin.     

Acute Babesia bovis infections: renal involvement in the hypotensive syndrome.

Splenectomised calves in metabolism cages were infected with Babesia bovis. During the infection, urine samples were collected and analysed for electrolytes, proteins, kinin, and urinary kallikrein. During the later stages of the infection there were significant reductions in urinary volume, water intake, urinary kinin, kallikrein, and electrolytes. Proteinuria was detected from 3--8 days postinfection of which 15--20% was haemoglobin and most of the remainder was albumin (70--75%). Fibrin degradation products, fibrinogen-like products, and haptoglobin were not detected. Degeneration of cortical tubules was detected by histological studies. As these tubules produce urinary kallikrein it seems probable that diminished glomerular blood flow and hence glomerular filtration rate are due to decreased production of this enzyme.     

On the binding of dolichol by bovine liver supernatant

Experimental evidence is presented that a bovine liver pH 5.1 supernatant possesses binding capacity towards dolichol. Optimal binding is found at physiological pH and at 5 degrees C. At higher temperature the binding is drastically reduced. After binding, the labelled ligand cannot be chased by unlabelled dolichol. Scatchard analysis indicates a single class of binding sites (Bmax = 3.6 pmol/mg protein) with an apparent Kd of 1.8 X 10(-11) M. Only dolichol and dolichyl derivatives drastically reduce the binding phenomenon. The involvement of a protein-like structure is inferred from ammonium sulphate precipitation and proteolysis experiments. Exclusion chromatography and gel electrophoresis under nondenaturating conditions indicate a high molecular weight of the binding complex. Upon SDS electrophoresis, bound [3H]dolichol comigrates with a single protein band (Mr approximately equal to 25,000).     

The excretion of lactate dehydrogenase in human urine after ingestion of aspirin

1. Cells present in normal human urine contain 5-10% of the total lactate dehydrogenase excreted. The enzyme released from these cells by ultrasonication contained a distribution of isoenzymes similar to that found in the bulk of the urine and it is suggested that these cells are the main source of urinary lactate dehydrogenase. 2. Cells were thoroughly washed before examination so it is unlikely that the enzyme found in urinary sediment was simply adsorbed. In addition, full recoveries of added lactate dehydrogenase isoenzymes LDH(1) and LDH(5) showed that adsorption did not occur. 3. Most of the cells in normal urine are of the non-squamous epithelial type and their excretion is greatly increased after the ingestion by the subject of 3g. of aspirin. The possible origin of these non-squamous cells from the kidney is discussed. 4. Starch-block electrophoresis and relative activity measurements of lactate dehydrogenase excreted after the subject had taken aspirin show that the enzymes present in urine and cells are very similar, confirming the conclusion reached above (point 1). They have slightly more M subunits than the normal, shown particularly as an increase in isoenzyme LDH(2). The isoenzyme pattern is like that of the kidney medulla and the possible reasons for this are discussed in terms of the concentration of salicylic acid in various parts of the kidney. 5. The results confirm the previous suggestion that the kidney is the main source of urinary lactate dehydrogenase.     

Atrial natriuretic factor inhibits vasopressin secretion in conscious sheep

To test the hypothesis that atrial natriuretic factor (ANF) has a centrally mediated action on body fluid homeostasis, the effects of intracerebroventricularly (ICV) infused ANF on plasma vasopressin (AVP) concentration and urinary water and electrolyte excretion were investigated in euhydrated and water-deprived conscious sheep. ICV ANF decreased plasma AVP concentration and increased urinary free water excretion in euhydrated sheep, with excretion of Na and K unaltered. However, ICV ANF did not affect urinary volume, free water clearance, or excretion of Na and K in dehydrated animals, although plasma AVP concentration was significantly decreased. The relationship between urine volume and plasma AVP concentration was fitted by a power curve: urine volume = 0.79 X [AVP]-0.71; urine volume changes very little as a function of AVP concentration at the higher ranges. Intravenous infusion of the same amount of ANF was without effect on plasma AVP concentration or urinary excretion in both euhydrated and dehydrated animals. Mean arterial pressure was unchanged throughout all experiments. These results are consistent with the hypothesis that central ANF inhibits AVP secretion.     

Relationship of urinary kallikrein excretion to urinary potassium excretion during furosemide administration with and without amiloride

The relationship of urinary kallikrein excretion to urine volume, and to urinary sodium and potassium excretions was studied in normal rats during furosemide diuresis and superimposed injection of amiloride, a K+-sparing diuretic. Continuous infusion of furosemide increased urinary kallikrein, sodium and potassium excretions and the urine volume. Amiloride injection during furosemide diuresis caused further increase in diuresis and natriuresis, but a prompt decrease in urinary kallikrein excretion to basal level, and potassium excretion to below the basal level. The significant correlation of urinary kallikrein excretion to urinary potassium excretion, but not to urine volume and urinary sodium excretion after amiloride injection suggests that the major determinant of urinary kallikrein excretion is renal potassium secretion through a mechanism that is affected by amiloride.     

Separation of dolichol and dolichyl-P in microsomal and lysosomal compartments of hepatocytes

The distribution, labeling and interrelationship of microsomal and lysosomal dolichol and dolichyl-P in rat liver was investigated. After membrane induction with phenobarbital, N-nitrosodiethylamine and diethylhexylphthalate, the amount of microsomal and lysosomal dolichols are modulated independently. Liposomal labeled dolichol injected into the portal vein appears only in lysosomes and even after 8 days is still limited to the lysosomes. After in vivo labeling with [3H]mevalonate, high initial labeling of dolichol and dolichyl-P is present in microsomes and the labeling in microsomes is greater than that in lysosomes even after 8 h. The results demonstrate compartmentalization of the intracellular dolichols in hepatocytes. These lipids may have independent roles at different membrane locations.     

Enhanced production of dolichol,but not dolichyl phosphate,in the earliest stages of rat liver regeneration

The regenerating liver presents a changed ability to use mevalonate 16 hr after partial hepatectomy. The dolichol content and its synthesis from mevalonate is increased, while no variation of dolichyl-P and ubiquinone parameters are detectable.The greater amount ofmevalonate utilized to form dolichol, but not dolichyl-P, in this proliferating system, raises some questions about the physiological significance of these isoprenoid compounds and about their biosynthetic sequence.     

Characterization of the reaction of GDP-mannose with dolichol phosphate in liver membranes.

The Mn-2+ dependent mannosyl transfer reaction between GDP-[14-C]mannose and dolichol phosphate, which is catalyzed by liver membranes, could not be followed accurately with the existing assay systems. Thus, GDP-[14-C]mannose is hydrolyzed rapidly by a pyrophosphatase present in microsomal and Golgi fractions from liver cells. The rate of the hydrolysis is rapid enough to limit the extent of incorporation of [14-c]mannose into endogenous acceptors. AMP was an effective inhibitor of the pyrophosphatase in Golgi membranes, and protected GDP-mannose from metabolism in alternative pathways. In the presence of AMP it was possible accurately to follow the time course of synthesis of dolichol phosphate [14-c]mannose over short time periods. Even though the time course of the reaction was measured over 2 s intervals, no linear portion could be detected in plots of product formed versus time. The kinetics of synthesis did, however, fit an equation for a first-order kinetic process. The basis for the first-order kinetics seems related to the very small amounts of dolichol phosphate in membranes. The values of the first-order rate constant is dependent on the concentrations of GDP-mannose and Mn-2+ added to the assays.     

Increased nonsterol isoprenoids, dolichol and ubiquinone, in the Smith-Lemli-Opitz syndrome: effects of dietary cholesterol

Smith-Lemli-Opitz syndrome (SLOS) is an inherited autosomal recessive cholesterol deficiency disorder. Our studies have shown that in SLOS children, urinary mevalonate excretion is normal and reflects hepatic HMG-CoA reductase activity but not ultimate sterol synthesis. Hence, we hypothesized that in SLOS there may be increased diversion of mevalonate to nonsterol isoprenoid synthesis. To test our hypothesis, we measured urinary dolichol and ubiquinone, two nonsterol isoprenoids, in 16 children with SLOS and 15 controls, all fed a low-cholesterol diet. The urinary excretion of both dolichol (P < 0.002) and ubiquinone (P < 0.02) in SLOS children was 7-fold higher than in control children, whereas mevalonate excretion was comparable. In a subset of 12 SLOS children, a high-cholesterol diet decreased urinary mevalonate excretion by 61% (P < 0.001), dolichol by 70% (P < 0.001), and ubiquinone by 67% (P < 0.03). Our hypothesis that in SLOS children, normal urinary mevalonate excretion results from increased diversion of mevalonate into the production of nonsterol isoprenoids is supported. Dietary cholesterol supplementation reduced urinary mevalonate and nonsterol isoprenoid excretion but did not change the relative ratios of their excretion. Therefore, in SLOS, a secondary peripheral regulation of isoprenoid synthesis may be stimulated.     

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual.