Effects of 4'-modified analogs of aristeromycin on the metabolism of S-adenosyl-L-homocysteine in murine L929 cells

(1'R,2'S,3')-9-(2',3'-Dihydroxycyclopentan-1'-yl)adenine (DHCaA), (1'R,2'S,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)-3-deazaadenine (3-deaza-DHCaA), (4'R)-4'-methyl-DHCaA, and (4'R)-4'-vinyl-DHCaA, which are analogs of the carbocyclic nucleoside aristeromycin, were synthesized earlier by our laboratory and were shown to be potent inhibitors of purified bovine liver S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1). In the present study, these analogs were shown to produce rapid (within 15 min) and concentration-dependent (0.03-10 microM) inhibition of AdoHcy hydrolase in cultured murine L929 cells [relative order of inhibitory activity, DHCaA = 3-deaza-DHCaA > (4'R)-4'-vinyl-DHCaA = (4'R)-4'-methyl-DHCaA]. The relative potencies of these inhibitors on the L929 AdoHcy hydrolase were consistent with their inhibitory effects on the recombinant forms of rat liver and human placental enzymes. This inhibition of L929 cellular AdoHcy hydrolase persisted for up to 48 hr. The inhibition of the L929 AdoHcy hydrolase resulted in a significant increase in the cellular concentrations of AdoHcy, whereas the cellular S-adenosylmethionine (AdoMet) levels remained relatively constant, thereby elevating the AdoHcy/AdoMet ratios. Maximum increases in AdoHcy levels and AdoHcy/AdoMet ratios occurred within 6 hr of exposure to the inhibitors and persisted for at least 24 hr. At a concentration of 1 microM, DHCaA and 3-deaza-DHCaA increased AdoHcy/AdoMet ratios to approximately 0.8 (after 24 hr of exposure to the inhibitors), whereas (4'R)-4'-vinyl-DHCaA and (4'R)-4'-methyl-DHCaA elevated AdoHcy/AdoMet ratios to approximately 0.15, compared with control levels of 0.05. Treatment of L929 cells with concentrations of DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA up to 10 microM did not result in changes in cellular levels of endogenous nucleotides (e.g., CTP, UTP, ATP, and GTP). In contrast, cells treated with 10 microM aristeromycin for 6 hr contained reduced cellular levels of CTP, ATP, and GTP and significant levels of aristeromycin triphosphate and a GTP metabolite of this carbocyclic nucleoside. These data clearly show that the 4'-modified analogs [DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA] retain inhibitory activity toward cellular AdoHcy hydrolase, causing elevated levels of AdoHcy and elevated AdoHcy/AdoMet ratios. However, these analogs are devoid of substrate or inhibitory activity toward cellular adenosine kinase. In addition, aristeromycin is rapidly metabolized in murine L929 cell lysates, i.e., > 60% of the aristeromycin had been metabolized in 6 hr. In contrast, neither DHCaA nor 3-deaza-DHCaA showed any decrease in concentration after incubation with cell lysates for up to 6 hr.     

Carbocyclic adenosine analogues as S-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances

Various carbocyclic analogues of adenosine, including aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, neplanocin A and 3-deazaneplanocin A, and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A have been recognized as potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. This enzyme plays a key role in methylation reactions depending on S-adenosylmethionine (AdoMet) as methyl donor. AdoHcy hydrolase inhibitors have been shown to exert broad-spectrum antiviral activity against pox-, paramyxo-, rhabdo-, filo-, bunya-, arena-, and reoviruses. They also interfere with the replication of human immunodeficiency virus through inhibition of the Tat transactivation process.     

Moderate amplifications of the c-myc gene correlate with molecular and clinicopathological parameters in colorectal cancer

Advanced glycation end product (AGE) formation is related to hyperglycemia in diabetes but not in uremia, because plasma AGE levels do not differ between diabetic and nondiabetic hemodialysis patients. The mechanism of this phenomenon remains elusive. Previously, it was suggested that elevation of AGE levels in uremia might result from the accumulation of unknown AGE precursors. The present study evaluates the in vitro generation of pentosidine, a well identified AGE structure. Plasma samples from healthy subjects and nondiabetic hemodialysis patients were incubated under air for several weeks. Pentosidine levels were determined at intervals by HPLC assay. Pentosidine rose to a much larger extent in uremic than in control plasma. Pentosidine yield, i.e., the change in pentosidine level between 0 and 4 wk divided by 28 d, averaged 0.172 nmol/ml per d in uremic versus 0.072 nmol/ml per d in control plasma (P < 0.01). The difference in pentosidine yield between uremic and control plasma was maintained in samples ultrafiltrated through a filter with a 5000-Da cutoff value and fortified with human serum albumin (0.099 versus 0.064 nmol/ml per d; P < 0.05). Pentosidine yield was higher in pre- than in postdialysis plasma samples (0.223 versus 0.153 nmol/ml per d; P < 0.05). These results suggest that a large fraction of the pentosidine precursors accumulated in uremic plasma have a lower than 5000 Da molecular weight. Addition of aminoguanidine and OPB-9195, which inhibit the Maillard reaction, lowered pentosidine yield in both uremic and control plasma. When ultrafiltrated plasma was exposed to 2,4-dinitrophenylhydrazine, the yield of hydrazones, formed by interaction with carbonyl groups, was markedly higher in uremic than in control plasma. These observations strongly suggest that the pentosidine precursors accumulated in uremic plasma are carbonyl compounds. These precursors are unrelated to glucose or ascorbic acid, whose concentration is either normal or lowered in uremic plasma. They are also unrelated to 3-deoxyglucosone, a glucose-derived dicarbonyl compound whose level is raised in uremic plasma: Its addition to normal plasma fails to increase pentosidine yield. This study reports an elevated level of reactive carbonyl compounds ("carbonyl stress") in uremic plasma. Most have a lower than 5000 Da molecular weight and are thus partly removed by hemodialysis. Their effect on pentosidine generation can be inhibited by aminoguanidine or OPB-9195. Carbonyl stress might contribute to AGE modification of proteins and thus to clinically relevant complications of uremia.     

Adverse effects of hyperhomocysteinemia and their management by folic acid

A moderate increase in plasma homocysteine is an independent risk factor for cardiovascular disease. Plasma homocysteine is frequently elevated in chronic renal failure and in uremic patients, and the major causes of death in these patients are cardiovascular accidents. Homocysteine metabolism and mechanisms of toxicity are reviewed. Homocysteine elevation in blood leads to the intracellular increase of its precursor, adenosylhomocysteine, a powerful inhibitor of adenosylmethionine-dependent transmethylations. In vitro evidence shows that this increase is reversible upon homocysteine removal. Membrane protein methylation levels are consistently reduced in erythrocytes of both chronic renal failure and hemodialysis patients. This widespread enzymatic methylation is a key step for the repair of molecular damage resulting from the spontaneous deamidation and isomerization reactions of susceptible residues in proteins. In agreement with these findings is the observation that the concentration of a stable side product, D-Asx, of the repair process is significantly lower in erythrocyte membrane proteins from hemodialysis patients than from controls, showing that the repair of damaged membrane proteins is actually defective. It has been shown that treatment with folates dramatically lowers plasma homocysteine, presumably by improving remethylation to methionine. This indicates that folates and/ or their active derivative, i.e., methyltetrahydrofolate, could be effective in ameliorating transmethylations as well.     

Autoxidation products of both carbohydrates and lipids are increased in uremic plasma: is there oxidative stress in uremia?

BACKGROUND: Advanced glycation end products (AGEs), formed by non-enzymatic glycation and oxidation (glycoxidation) reactions, have been implicated in the pathogenesis of several diseases, including normoglycemic uremia. AGE research in uremia has focused on the accumulation of carbohydrate-derived adducts generated by the Maillard reaction. Recent studies, however, have demonstrated that one AGE, the glycoxidation product carboxymethyllysine (CML), could be derived not only from carbohydrates but also from oxidation of polyunsaturated fatty acids in vitro, raising the possibility that both carbohydrate and lipid autoxidation might be increased in uremia. METHODS: To address this hypothesis, we applied gas chromatography-mass spectrometry and high performance liquid chromatography to measure protein adducts formed in uremic plasma by reactions between carbonyl compounds and protein amino groups: pentosidine derived from carbohydrate-derived carbonyls, malondialdehyde (MDA)-lysine derived from lipid-derived carbonyls, and CML originating possibly from both sources. RESULTS: All three adducts were elevated in uremic plasma. Plasma CML levels were mainly (>95%) albumin bound. Their levels were not correlated with fructoselysine levels and were similar in diabetic and non-diabetic patients on hemodialysis, indicating that their increase was not driven by glucose. Pentosidine and MDA-lysine were also increased in plasma to the same extent in diabetic and non-diabetic hemodialysis patients. Statistical analysis indicated that plasma levels of CML correlated weakly (P < 0.05) with those of pentosidine and MDA-lysine, but that pentosidine and MDA-lysine varied independently (P > 0.5). CONCLUSIONS: These data suggest that the increased levels of AGEs in blood, and probably in tissues, reported in uremia implicate a broad derangement in non-enzymatic biochemistry involving alterations in autoxidation of both carbohydrates and lipids.     

Medium-sized peptides in the blood of patients with uremia

We have carried out high performance liquid chromatographic analysis of serum and ultrafiltrate of blood obtained from uremic patients and normal subjects to elucidate the presence of medium-sized peptides unique to uremia. Many fluorescamine-positive substances, excluding amino acids and guanidine compounds, were increased in uremic serum compared with normal serum. At the 0.5-1.0 pmol/ml serum level, several peaks were unique to uremia. The retention time, fluorescamine reactivity, molecular weight distribution and the result of enzymatic digestion revealed that these peaks are peptidic substances.     

RNA metabolism in uremic patients: accumulation of modified ribonucleosides in uremic serum. Technical note

To determine the metabolism of ribonucleic acid (RNA) in uremia, serum and urine levels of ribonucleosides in uremic patients were analyzed using reversed-phase high-performance liquid chromatography. The serum levels of xanthosine and all modified ribonucleosides were increased in undialyzed patients with chronic renal failure (CRF), and patients undergoing hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD). The serum level of pseudouridine was markedly increased in all the uremic patients especially CAPD patients (32 times higher than normal). By contrast, the serum level of adenosine did not show any significant change in the uremic patients. Interestingly, the serum and urine levels of inosine were significantly decreased in all the uremic patients, suggesting that the production of inosine is decreased in uremic patients. The serum level of uridine was significantly elevated only in the HD patients. The serum levels of all ribonucleosides except inosine and uridine decreased significantly after HD. The urinary excretion of inosine, 1-methyladenosine, 1-methylguanosine, N2,N2-dimethylguanosine and N4-acetylcytidine was significantly decreased in the CRF patients, leading to the accumulation of these modified ribonucleosides in the uremic serum. CAPD patients showed markedly increased serum levels of modified ribonucleosides such as pseudouridine, 1-methylinosine, and N2,N2-dimethylguanosine and N4-acetylcytidine as compared with the HD patients. These results demonstrate that there was an altered metabolism of RNA in uremic patients with marked accumulation of modified ribonucleosides.     

Comparison of the thiamine level in blood and erythrocyte transketolase activity in hemodialyzed and nondialyzed patients during recombinant human erythropoietin therapy

Thiamine and erythrocyte transketolase activity (ETKA) disturbances in end-stage renal disease are caused mainly by uremia and dialysis treatment. We examined whether recombinant human erythropoietin (rhEPO) can correct these abnormalities in uremic patients. Thirteen hemodialysis (HD) and 12 nondialyzed (ND) anemic patients showed decreased free and total thiamine levels in plasma and in erythrocytes and decreased ETKA when compared to 20 healthy subjects. Thiamine blood levels (mumol/l) were determined using a fluorimetric technique, and ETKA (mumol/l per minute) was assessed with a photocolorimetric method. Over 20 weeks of study, rhEPO was given intravenously for 8 weeks at 50 Ul/kg body weight (BW) three times a week, and subcutaneously for 4 weeks at 25 Ul/kg BW, twice a week, and for the last 8 weeks at 25 Ul/kg BW once a week. The correction of anemia was associated with an increase in plasma thiamine and erythrocyte total thiamine as well as ETKA in HD patients and with an increase in erythrocyte total thiamine in ND patients only during the period of intravenous infusions.     

Effects of ions on adenosine binding and enzyme activity of purified S-adenosylhomocysteine hydrolase from bovine kidney

The present investigation was undertaken to determine the effect of various ions on the characteristics of S-adenosylhomocysteine (SAH) hydrolase from bovine kidney. The binding sites of [3H]-adenosine to purified SAH hydrolase were not influenced by phosphate, magnesium, potassium, sodium, chloride or calcium ions at physiological cytosolic concentrations. To test whether NAD+ in the SAH hydrolase is essential for adenosine binding, we prepared the apoenzyme by removing NAD+ with ammonium sulfate. The resulting apoenzyme did not exhibit any [3H]-adenosine binding. Since the apoenzyme was enzymatically inactive, it is suggested that adenosine binds to the active site and not to an allosteric site of the intact enzyme. The kinetics of the hydrolysis and the synthesis of SAH catalyzed by the enzyme SAH hydrolase were measured in the presence and absence of phosphate and magnesium. Phosphate increased the Vmax for both synthesis and hydrolysis. However, only the affinity of adenosine for SAH synthesis was significantly enhanced from 10.1+/-1.3 microM to 5.4+/-0.5 microM by phosphate. This effect was already maximal at a phosphate concentration of 1 mM. All other tested ions were without effect on the enzyme activity. Our results show that phosphate at physiological concentrations shifts the thermodynamic equilibrium of SAH hydrolase in the direction of SAH synthesis. These findings imply that SAH-sensitive transmethylation reactions are inhibited during renal hypoxia when intracellular levels of phosphate, adenosine, and SAH are elevated.     

Triphenyltin chloride induces glucose intolerance by the suppression of insulin release from hamster pancreatic beta-cells

An increased activity of phospholipase A2 has been observed in the plasma of patients with uremia. This enzyme converts phosphatidylcholine to lysophosphatidylcholine (LPC), an inhibitor of platelet aggregation. We measured the levels of plasma phospholipids including LPC, and platelet aggregation in 7 patients with uremia. Platelet response to agonists was defective, mainly with collagen (p < 0.001). The patients' levels of LPC in plasma were similar to those of controls (109.7 +/- 41.6 vs. 80.4 +/- 16.8 nmol/ml) and did not correlate with the platelet response to adenosine diphosphate (r = -0.51). The amount of phosphatidylcholine was increased with respect to normal plasma (1,041.0 +/- 201.8 vs. 760.8 +/- 142.7 nmol/ml, p < 0.01), while the levels of other phospholipids were normal. These results do not suggest a participation of plasma LPC in the genesis of the platelet defect observed in patients with uremia.     

Intracellular concentrations of mitoxantrone in leukemic cells in vitro vs in vivo

The aim of this study was to determine the intracellular pharmacokinetics of mitoxantrone in vivo and to use these results to establish how leukemic cells should be incubated to perform clinically relevant in vitro studies of this drug. Blood samples were obtained from 11 patients with acute nonlymphoblastic leukemia at certain intervals up to 20 h after the infusion of mitoxantrone 12 mg/m2. Plasma and leukemic cells were separated and the drug concentrations were determined with HPLC. Before treatment, leukemic cells from 12 patients were incubated with 0.02, 0.05, 0.1, 0.2 and 1.0 microM mitoxantrone for 1-4 h and thereafter cultured in suspension culture for 20 h; during this time cell samples were taken at certain intervals for drug determination. In cells incubated with 0.05 and 0.2 microM mitoxantrone the cytotoxic effect was measured with the DiSC assay after cultivation for 4-5 days. In vivo, the intracellular levels exceeded the plasma concentrations already at the end of infusion and after 2 h the intracellular concentrations were 200-300 times higher than in plasma. In vitro, the intracellular steady state level of mitoxantrone was reached after 1-2 h and there was a pronounced intracellular retention even after 20 h culture in drug-free medium. Incubation with 0.05 microM during 1 h gave intracellular concentrations of mitoxantrone similar to those achieved in vivo. This incubation concentration gave a mean cytotoxic effect of 53% living cells measured with the DiSC assay, which gives good possibilities to discriminate between mitoxantrone-sensitive and unsensitive cells. We believe that exposing leukemic cells in vitro for in vivo mimicking mitoxantrone concentrations could increase the clinical relevance of predictive assays.     

Hyperhomocysteinemia is associated with atherosclerotic occlusive arterial accidents in predialysis chronic renal failure patients

Hyperhomocysteinemia has been shown to constitute an independent risk factor for premature occlusive arterial disease. Moderate hyperhomocysteinemia is present in chronic uremic patients, who often develop premature atherosclerosis, but no direct evidence of an association between the occurrence of atherosclerotic cardiovascular accidents (CVAs) and hyperhomocysteinemia has yet been reported in such patients. We serially determined total plasma homocysteine (Hcy) levels in a cohort of 93 consecutive chronic renal failure, undialyzed patients (57 males, 36 females) with creatinine clearance (Ccr) < 50 ml/min.1.73 m2 and age > or = 50 years at start of follow-up, together with serial assessment of Ccr and blood lipid parameters. From January 1989 to December 1995, 24 patients (group 1) experienced myocardial infarction (18 cases, 13 males) or cerebral infarction (6 cases, 3 males) while the remaining 69 (group 2) remained free of CVAs. Patients in groups 1 and 2 did not differ with respect to age (66 +/- 1.8 vs. 65 +/- 1.1 years, mean +/- Se) or serum creatinine (227 +/- 24 vs. 251 +/- 36 mumol/l) at onset of a CVA (group 1) or at the end of follow-up (group 2). The mean Hcy level was significantly higher in group 1 (20.7 +/- 1.6 vs. 12.8 +/- 0.5 mumol/l, p < 0.0001), as was the proportion of patients with Hcy in excess of 14 mumol/l, the upper limit in healthy controls (83 vs. 30%, p < 0.0001). Logistic regression analysis identified Hcy as an independent risk factor for CVA, with an odds ratio of 11.4 (95% confidence interval 3.5-37.7), which remained significant after adjustment on other variables. We conclude that an elevated Hcy level is associated with a risk of occlusive arterial accidents in patients with chronic renal failure and that hyperhomocysteinemia contributes to the accelerated atherosclerosis complicating chronic uremia.     

The roles of renal catabolism and uremia in modifying the clearance of fibrinogen and its degradative fragments D and E

Elevated levels of fibrinogen/fibrin degradation products (FDP) occur in uremia, and have been thought to be in part related to intravascular coagulation in the kidney. More recent data indicated that delayed catabolism of fibrinogen fragment D occurred in anephric animals. To further evaluate FDP catabolism in the kidney, turnover studies of purified dog 131I-Fg-D and 125I-Fg-E were performed on dogs before and after acute subtotal nephrectomies, and later during chronic uremia. 131I-fibrinogen clearances were also perfomed. Slowed catabolism of Fg-D and Fg-E was observed in both the acute and chronic uremic stages. Altered urinary excretion was not a factor as only minimal amounts of Fg-D and Fg-E were excreted in the urine of the control animals. In the 131I-fibrinogen studies, there were significant changes in plasma volume, fibrinogen t 1/2, and intravascular/extravascular distribution, but not in fractional catabolic rate. To differentiate fully, the effects of uremia from those of loss of catabolic renal tissue, the Fg-D and Fg-E turnover studies were repeated on other animals with intact kidneys whose ureters were diverted into the peritoneum and compared to subsequent studies after total nephrectomy. The control and ureter-severed studies had the same clearance pattern, whereas decreased catabolism occurred in the nephrectomized dogs. The results demonstrate uremia per se does not have a major effect upon the catabolism of fibrinogen, Fg-D, and Fg-E. Loss of renal tissue does impair the clearance of Fg-D and Fg-E, indicating these proteins are normally catabolized in part by the kidneys. Thus elevated plasma FRA in uremic patients may reflect decreased Fg-D and Fg-E catabolism rather than increased FDP production from primary or secondary fibrinolysis.     

Increased lipase inhibition in uremia: identification of pre-beta-HDL as a major inhibitor in normal and uremic plasma

The hypertriglyceridemia commonly observed in uremia has been attributed to an abnormally high inhibitor activity in plasma for lipoprotein lipase (LPL) and hepatic lipase (HL), both of which have a key role in lipoprotein metabolism. The purpose of this investigation was to establish a relationship between plasma lipase inhibitor activity and hypertriglyceridemia, identify the main plasma lipase inhibitor, and determine the basis for the greater inhibitor activity in uremia. In a mixed population of normal (N = 8) and uremic subjects (N = 12), log-transformed plasma triglycerides correlated with both inhibitor activity and uremic status. However, inhibitor activity was the only retained predictor variable for triglycerides in a multiple linear regression model (r = 0.91; P < 0.0001). An inhibitor isolated from normal plasma was identified as a particle containing apolipoprotein A-I (apo A-I) and 3% phospholipid. This particle, which has pre-beta electrophoretic mobility and a Stokes' radius of 54 A, therefore corresponds to a form of the previously described pre-beta-HDL (free apo A-I) in the non-lipoprotein fraction of plasma. Comparison of normal and uremic plasma indicated that the greater lipase inhibitor activity in the latter could be attributed to an increased concentration of apo A-I in the non-lipoprotein fraction of plasma (pre-beta-HDL), as well as to increased inhibition by the uremic lipoproteins. The increased plasma lipase inhibitor activity may be important in the pathogenesis of hypertriglyceridemia in chronic renal failure.     

Involvement of P1 receptors in the effect of forskolin on cyclic AMP accumulation and export in PC12 cells

In PC12 cells, forskolin as well as the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased intracellular adenosine-3',5'-cyclic monophosphate (cyclic AMP) levels, which peaked at 45-60 minutes and declined thereafter. Maximum levels were 3000 and 1700 pmol/10(6) cells during treatment with 10 microM forskolin or 0.1 microM NECA, respectively. Extracellular cyclic AMP rose with time, at mean rates of 24.7 (forskolin) and 11.3 (NECA) pmol/min/10(6) cells. With either drug, a linear correlation was obtained between the calculated time integral of intracellular cyclic AMP and the measured extracellular cyclic AMP levels, indicating that the outflow of cyclic AMP was sustained by a nonsaturated transport system. The ability of forskolin to increase intracellular and extracellular cyclic AMP levels was hindered in a concentration-dependent manner by 8-(p-sulfophenyl)theophylline (8-SPT). A similar inhibition was exerted by other two adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine. The concentration-response curve to adenosine was shifted to the right by 25 microM 8-SPT, whereas that of forskolin was shifted downwards. Adenosine deaminase (ADA, EC 3.5.44, 1 U/mL) reduced the intracellular cyclic AMP response to forskolin by 68%, whereas the adenosine transport inhibitor, dipyridamole (10 microM), significantly increased 1 and 10 microM forskolin-dependent cyclic AMP accumulation. Erythro-9-(2-hydroxy-3-nonyl)adenine (10 microM), an inhibitor of ADA, and alpha,beta-methyleneadenosine 5'-diphosphate (100 microM), an inhibitor of ecto-5'-nucleotidase, did not alter forskolin activity. These results demonstrate that a cyclic AMP extrusion system operates in PC12 cells during adenylyl cyclase stimulation by forskolin and that this stimulation involves a synergistic interaction with endogenous adenosine. However, extruded cyclic AMP does not appear to significantly contribute to the formation of the endogenous adenosine pool.     

Determination of trace lithium in human erythrocytes by electrothermal atomic-absorption spectrometry with pyrocoated graphite tubes and integrated platform

Electrothermal graphite-furnace atomic-absorption spectroscopy with pyrocoated graphite tubes, integrated platform and matrix modification was used to determine submicromolar concentrations of trace lithium in human red blood cells. Matrix-matched samples were used to establish calibration curves for concentrations up to 0.58 microM (addition-calibration method) with satisfactory linearity (r2 > 0.99) and intra- and inter-day variability (CV < 11.4%). The median concentration of trace lithium in the cells of 40 healthy Caucasian volunteers devoid of medical or psychiatric history was 0.23 microM (inter-quartile range 0.20-0.30). The levels of trace lithium in the red blood cells correlated (r2 = 0.83) with plasma concentrations (median 0.13 microM, inter-quartile range 0.11-0.19) measured in the same blood sample. Dietary factors (e.g. consumption of lithium-containing mineral water) affected both levels. The red blood cell/plasma lithium ratio had a median value of 1.57 (inter-quartile range 1.16-2.07), implying that trace lithium is accumulated in erythrocytes. This contrasts with most reports of red blood cell/plasma ratio, measured during therapeutic treatment with lithium, for which the average value is 0.5-0.8, albeit for much higher concentrations of lithium (approx. 500-800 microM). The proposed analytical method has the required sensitivity and accuracy for determination of trace lithium in red blood cells and makes it possible to perform epidemiological studies to assess human exposure to environmental lithium in diet and beverages, and inter-individual variations in trans-membrane and renal lithium kinetics at the submicromolar level.     

Determination of tin in food products using pyrocatechol violet

The practice of dietetic therapy is unusual today for patients suffering from renal failure without hypertension and reduction of glomerular filtration rate. Specific treatment is needed, however, for arterial hypertension, uremia, calculus and uralith disease. Experiments in rats showed, that a lot of uremic symptoms following poorly functioning kidneys are partly at least caused by disturbances in amino acid metabolism. Uremia patients with dysfunctioning plasma protein metabolism (transferrin, complement, cholinesterase, prealbumin and retinolbinding protein) need oral, respectively parenteral substitution of essential amino acids. This substitution is very important under catabolic stress conditions in uremic syndrome with and without vividialysis treatment.     

Plasma catecholamines, neuropeptide Y and leucine-enkephalin in uremic patients before and after dialysis during rest and handgrip

We have studied the effects of handgrip on plasma levels of catecholamines, neuropeptide Y (NPY), and leu-enkephalin before and after hemodialysis of uremic patients. A cuprophan dialyzer was used. We found, that dopamine level was higher in uremia group before hemodialysis both during rest (0.38 +/- 0.39 pmol/ml) and handgrip (1.13 +/- 1.00 pmol/ml) compared to control (0.17 +/- 0.19, and 0.66 +/- 0.83 pmol/ml respectively). Hemodialysis leads to further increase of its level (0.49 +/- 0.35 pmol/ml) at rest. Epinephrine level was almost the same in uremic patients before (0.43 +/- 0.51 pmol/ml) and after dialysis (0.46 +/- 0.60) as in control subjects (0.41 +/- 0.37 pmol/ml) during the rest. Its level measured after the handgrip was the highest in uremic group after dialysis (2.10 +/- 2.00 pmol/ml), significantly lower before dialysis (1.26 +/- 0.85 pmol/ml), and the lowest in control group (0.78 +/- 0.43 pmol/ml). Norepinephrine level were very similar in uremic group before dialysis (1.54 +/- 1.05 pmol/ml), after dialysis (1.79 +/- 1.29 pmol/ml) and in control group (1.46 +/- 1.06 pmol/ml) during the rest. During the handgrip test its level was higher in uremic group after hemodialysis than before it (adequate values 8.78 +/- 4.61 and 6.70 +/- 4.74 pmol/ml). The difference between uremia group before dialysis and control group did not reach significance. The level of NPY has the tendency to increase in uremic patients. Dialysis leads to following increase of its level, but the changes did not reach the significance both in rest and handgrip. Leu-enkephalin level was higher in uremic group (9.21 +/- 7.62 pmol/ml) compared to control (5.22 +/- 1.53 pmol/ml). We observed non-significant fall of this level after dialysis (6.79 +/- 4.76 pmol/ml). We found the same tendency during the handgrip, and the changes were significant. In conclusion: uremia per se leads to increase the level of dopamine and leu-enkephaline during the rest and handgrip, but the level of epinephrine only during the handgrip; dialysis leads to further increase of dopamine during the rest, but epinephrine, norepinephrine and leu-enkephaline only during the handgrip.