Electrophysiological brainstem investigations in obstructive sleep apnoea syndrome

In children with steroid-resistant nephrotic syndrome (SRNS) hyperlipidaemia may in the long term be associated with progressive renal insufficiency and increased risk of coronary heart disease. We have assessed the efficacy and tolerability of diet prior to and in combination with a hydroxymethylglutaryl CoA reductase inhibitor, simvastatin, in seven children with SRNS with a mean age of 8 years (range 1.8-16.3 years). Dietary advice to maintain adequate energy and protein intakes with reduced saturated fat and cholesterol intake had little impact on lipid levels pre treatment (mean reduction in cholesterol 1 mmol/l, triglyceride 1.1 mmol/l) but was maintained throughout the study duration. The mean cholesterol and triglyceride concentrations pre treatment were 12.1 +/- 2 (SEM) mmol/l and 8 +/- 2.1 (SEM) mmol/l, respectively. On a median simvastatin dose of 10 mg/day (range 5-40 mg) there was a 41% reduction in cholesterol to 6.6 +/- 0.77 (SEM) mmol/l and a 44% reduction in triglyceride to 3.9 +/- 1.38 (SEM) mmol/l at 6 months which was sustained at 12 months in five patients. The drug was well tolerated with no clinical side effects being noted. Over 6 months the mean plasma albumin concentrations increased from 18.2 +/- 1.26 (SEM) g/l to 23 +/- 2.51 (SEM) g/l, accounted for by three patients (1 complete remission, 1 partial remission, 1 end-stage renal failure). Plasma creatinine concentrations remained stable in five patients with two having progressive chronic renal failure. Growth parameters for both weight and height were maintained. Simvastatin has a beneficial effect on abnormal lipid levels in SRNS but the effectiveness of long-term therapy needs to be evaluated.     

Binding properties of (+/-)[3H]benidipine hydrochloride to rat heart membranes

A simple routine method for the gas chromatographic determination of methylamine, dimethylamine, ethylamine and methylethylamine in urine is presented. The method is based on a two-phase derivatization procedure with isobutyl chloroformate as reagent. The reaction is quantitative in 10 min. We found no artifact formation of either choline or trimethylamine (dietary amine compounds) or of dimethylethylamine or triethylamine (catalyst amines in the industrial setting). The chromatographic behaviour of the amine carbamates was excellent. The recoveries of methylamine, dimethylamine, ethylamine and methylethylamine in spiked urine samples were 82, 89, 100 and 96%, respectively, and the precision (the relative standard deviation) was 3.6, 1.8, 3.3 and 2.0%, respectively. The method was linear for the studied amine carbamates up to 250 mg/l. The endogenous amine concentrations in urine samples from ten normal subjects were: methylamine, 0.9 mg/l (mean; range 0.3-1.5); dimethylamine, 14.7 mg/l (mean; range 4.6-27.6); ethylamine, 0.8 mg/l (mean; range 0.2-2.3); methylethylamine, less than 0.02 mg/l.     

The determination of inorganic sulphate in serum and synovial fluid by high performance ion chromatography

A method for the determination of inorganic sulphate based on high performance ion chromatography is presented. The separation was performed on an anion-exchange column with a 1.8 mmol/l sodium carbonate/ 1.7 mmol/l sodium hydrogen carbonate-buffer, pH 10.35. Conductivity of the eluate was monitored after suppression of the background conductivity caused by the eluent-buffer. Serum and synovial fluid samples were prepared by ultrafiltration through membranes with a molecular mass cutoff of M(r) 10,000. The viscosity of the synovial fluids was reduced by treatment with hyaluronate lyase before ultrafiltration. The method showed a linear response for sulphate concentrations between 0.5 and 1000 mumol/l. The limit of detection was 1 mumol/l for aqueous standards. For serum the coefficient of variation within-run was 2.3%-2.4%, the coefficient of variation between days 2.9%-3.1%. For synovial fluids the coefficient of variation within-run was 3.1%-3.4%, the coefficient of variation between days 4.6%-5.7%. Standard recovery experiments performed by spiking pools of human sera containing low sulphate concentrations with sulphate concentrations between 5 mumol/l and 40 mumol/l showed recoveries between 98.9% and 100.6%. The corresponding experiments with pools of synovial fluids showed recoveries of 98.3% to 100.9%. As determined from 127 serum samples the reference range for sulphate was 262 mumol/l-420 mumol/l, with a mean value of 314 mumol/l. No dependence on age or sex was observed. The sulphate concentration in 36 synovial fluids from knees affected by inflammatory processes showed a mean value of 424 mumol/l and a standard deviation of 70 mumol/l. In 41 synovial fluids from knees affected by chronic degeneration joint disease, the sulphate concentrations were statistically significantly lower, with a mean of 374 mumol/l and a standard deviation of 58 mumol/l. The concentrations of sulphate in the synovial fluids were statistically significantly higher than those in the serum samples used for determination of the reference range. Following the oral application of a subtoxic single dose of acetaminophen (32.5 mg/kg body weight-62.5 mg/kg body weight) to 4 healthy volunteers, there was a significant decrease in the concentration of sulphate in serum with a minimum at 4-5 h after application of the drug. The cumulative concentration decrease of sulphate in serum and the kinetic constant of the sulphate depletion were not correlated with the applied acetaminophen dose normalized for body weight.     

Direct determination of procainamide and N-acetylprocainamide by capillary zone electrophoresis in pharmaceutical formulations and urine

A method is described for the simultaneous determination of sixteen organochlorine pesticides in drinking water using automated solid-phase extraction followed by high-volume (80 microliters) capillary column gas chromatography using electron capture detection. The fully automated extraction method followed by high-volume injection permits rapid sample analysis compared to previously described procedures since no further pre-concentration of the analytes is necessary after they have been eluted from the octadecyl solid-phase extraction cartridge. The lowest detectable concentrations of the pesticides are between 1-5 ng l(-1), relative recoveries range from 92-105% in tap water spiked at 100 ng l(-1) and the relative standard deviations are in the range 5-12%.     

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithiocarbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 microl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150 x 3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 microg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 microg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 microg/ml in plasma (500 microl) using a 1/y weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ratio values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 microg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

Home blood glucose concentrations in maturity-onset diabetes

Blood glucose concentrations during normal daily activities were measured in 106 patients with maturity-onset diabetes from capillary blood samples collected on to filter paper. Samples were taken before and two hours after main meals, before going to bed, and, in 51 cases, during the night. Fasting and mid-morning values were closely correlated with the mean values over 24 hours irrespective of the type of anti-diabetic treatment being given. Postprandial blood glucose concentrations remained below 11.5 mmol/l (207 mg/100 ml) when the fasting blood glucose value was 7.0 mmol/l (126 mg/100 ml) or less, and repeated fasting blood glucose values exceeding 7.0 mmol/l were associated with raised blood glycosylated haemoglobin concentrations. Diabetic control in maturity-onset diabetes may be satisfactorily monitored by regular measurement of fasting or mid-morning blood glucose values.     

Cholesterol synthesis is increased in mixed hyperlipidaemia

We showed previously that hypertriglyceridaemia, but not hypercholesterolaemia, is correlated with increases in cholesterol synthesis and apolipoprotein B secretion in patients with secondary hypertriglyceridaemia. The aim of the present study was to compare the rate of cholesterol synthesis, using fasting plasma mevalonic acid (MVA) as an index, in patients with primary mixed hyperlipidaemia (type IIb phenotype, n=45) and primary hypercholesterolaemia (type IIa phenotype, n=92). LDL cholesterol was significantly higher in types IIa (6.38+/-0.18 mmol/l) and IIb (5.89+/-0.25 mmol/l) compared to 40 normolipidaemic controls (2. 99+/-0.1 mmol/l, P<0.0001), whereas serum triglyceride was higher in type IIb (2.62 (range 2.2-3.0) mmol/l) than type IIa (1.22 (range 0. 85-1.60) mmol/l, P<0.001) and controls (0.90 (range 0.68-1.24) mmol/l, P<0.001). Similarly, MVA was higher in type IIb (7.0+/-0.46 ng/ml) than IIa (5.6+/-0.23 ng/ml, P<0.0) and controls (5.6+/-0.36 ng/ml, P<0.05). Plasma MVA correlated positively with serum triglyceride (r=0.22, P=0.004) and negatively with LDL cholesterol (r=-0.21, P=0.014). These results are in accordance with previous observations that VLDL-apolipoprotein B secretion and cholesterol synthesis are linked and demonstrate that the latter is increased in mixed hyperlipidaemia.     

Long term metabolic effects of two dietary methods of treating hyperlipidaemia

OBJECTIVES: To compare the long term metabolic effects of two diets for treating hyperlipidaemia. DESIGN: Randomised controlled study: after three weeks of normal (control) diet, subjects were randomly allocated to one of two test diets and followed up for six months. SETTING: Lipid clinic of tertiary referral centre in Naples. SUBJECTS: 63 subjects with primary type IIa and IIb hyperlipoproteinaemia entered the study, and 44 completed it. Exclusion criteria were taking drugs known to influence lipid metabolism, evidence of cardiovascular disease, homozygous familial hypercholesterolaemia, and body mass index over 30. INTERVENTIONS: Two test diets with reduced saturated fat (8%) and cholesterol (approximately 200 mg/day): one was also low in total fat and rich in carbohydrate and fibre, and the other was low in carbohydrate and fibre and rich in polyunsaturated and monounsaturated fats. MAIN OUTCOME MEASURES: Fasting plasma lipid and lipoprotein concentrations; blood glucose, insulin, and triglyceride concentrations before and after a test meal. RESULTS: In comparison with the control diet, both test diets induced significant and similar decreases in low density lipoprotein cholesterol concentrations (by a mean of 0.72 (SE 0.15) mmol/l, P < 0.001, for low total fat diet; by 0.49 (0.18) mmol/l, P < 0.05, for high unsaturated fat diet) and plasma triglyceride concentrations (by 0.21 (0.09) mmol/l, P < 0.05, for low total fat diet; by 0.39 (0.15) mmol/l, P < 0.05, for high unsaturated fat diet), while high density lipoprotein cholesterol concentrations after fasting and plasma glucose and insulin concentrations during test meals were not modified by either diet. CONCLUSIONS: Both test diets are suitable (alone or in combination) for treatment of hypercholesterolaemia.     

Intra-individual variations in triglyceride and total cholesterol levels in serum in mixed hyperlipidemia

Swings in serum concentrations of total cholesterol and triglycerides were assessed retrospectively in 12 of 60 patients with mixed hyperlipidaemia (ten men, two women; mean age 44 [38-55] years). A second disease as a cause of the hyperlipidaemia had been excluded. Despite clinical stability and unchanged drug therapy the fasting values of triglycerides, measured enzymatically, in a minimum of four different samples (over several months) differed by more than 5 mmol/l, a mean of 16.6 (5-41.9) mmol/l. The lowest values were 2.7 (1.3-7.5) mmol/l, the highest 17.9 (6.4-44.6) mmol/l. Total cholesterol concentrations varied around 5.5 (1.6-31.7) mmol/l, minimal values 5.7 (4.2-8.9) mmol/l, maximal ones 12.0 (6.9-37.4) mmol/l. Six of the twelve patients consumed more than 60 g alcohol daily. The cause of the marked variations between individual samples is uncertain. Marked swings in triglyceride and total cholesterol concentrations are likely in mixed hyperlipidaemia. It is, therefore, essential to measure these concentrations repeatedly to assess correctly the diagnosis and treatment.     

Reproducibility of fasting serum cholesterol and triglycerides in ambulatory patients with mixed hyperlipidemia

Intraindividual variability of serum lipid concentrations in normal volunteers and in patients with hyperlipidemia is substantial. The aim of this study was to investigate prospectively the reproducibility of fasting serum triglyceride and total cholesterol concentrations in primary health care patients with combined hyperlipidemia, i.e. under conditions of daily medical practice. Secondary forms of hyperlipidemia were excluded. 19 general medical outpatients with primary combined hyperlipidemia were studied. Serum total cholesterol and triglyceride concentrations were measured after an overnight fast at 08.00 h 4 times at weekly intervals. To study the influence of alcohol intake on serum lipid concentrations, total cholesterol and triglycerides were measured without alcohol influence and 12 hours after consumption of a mean of 100 g alcohol in the evening. In 19 patients (10 males, 9 females, mean age 55 years, body mass index 27.9 +/- 4.4 kg/m2), mean +/- SD of serum triglycerides was 3.97 +/- 1.8 mmol/l and of total cholesterol 7.9 +/- 1.8 mmol/l. The combined intraindividual and interassay coefficient of variation was 18.7 +/- 8.2% for triglycerides and 5.1 +/- 2.5% for total cholesterol. Fasting serum triglycerides (3.5 +/- 1.1 vs. 3.7 +/- 1.4 mmol/l) and total cholesterol (7.6 +/- 1.4 vs. 7.8 +/- 1.0 mmol/l) did not significantly change 12 hours after acute alcohol consumption. Patients with primary combined hyperlipidemia in a primary health care setting show small intraindividual variations of overnight fasted serum triglyceride and total cholesterol concentrations. Moderate alcohol consumption 12 hours before blood sampling does not significantly affect triglyceride and cholesterol values.     

Integrated analysis of diffusion and relaxation of water in blood

A fully validated gas chromatographic-tandem mass spectrometric (GC-MS-MS) method is described for the accurate determination of acetylsalicylic acid (ASA) in human plasma after a single low-dose oral administration of aspirin or guaimesal, an ASA releasing prodrug. ASA and the newly prepared O-[2H3]-acetylsalicylic acid (d3-ASA) used as internal standard were determined in 100-microl aliquots of plasma by extractive pentafluorobenzyl (PFB) esterification using PFB bromide and tetrabutylammoniumhydrogen sulphate as the esterifying and ion-pairing agent, respectively, and by GC-MS-MS analysis in the negative-ion chemical ionization mode. The overall relative standard deviations were below 8% for ASA levels in the range 0-1 microg/ml plasma. Mean accuracy was 3.8% for ASA levels within the range 0-100 ng/ml. The limit of quantitation of the method was determined as 200 pg/ml ASA at an accuracy of 5.5% and a precision of 15.2%. The limit of detection was determined as 546 amol of ASA at a signal-to-noise ratio of 10:1.     

Comparison of plasma creatinine determined with the Greiner Selective Analyser GSA II and the glomerular filtration rate

1. Reference values for the plasma creatinine were established using the alkaline picrate method with the Greiner Selective Analyzer GSA II in relation to the Cr 51-EDTA Clearance. Individuals with normal GFR between 93 to 159 ml/min/1.73 m2 had creatinine values in men (n = 65) from 53.7 to 119.5 mumol/l (0.61 to 1.35 mg/100 ml) and in women (n = 59) from 37.7 to 107 mumol/l (0.42 to 121 mg/100 ml). 2. The creatine determinations with the GSA II were compared to those on the Technicon Analyzer, the Beckman Creatinine Analyzer, the Gemsaec-Fast Analyzer and to the enzymatic creatinine method. A good correlation (r = 0.9780-0.984) was observed. 3. With the GSA II and the enzymatic method, bilirubin showed a minor interference which was more marked with the Beckman analyzer.     

Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates

We have studied, in a prospective blinded fashion, the effects of regular and extended-release gemfibrozil on plasma lipoprotein and apolipoprotein (apo) levels in hypercholesterolemic subjects with decreased high density lipoprotein (HDL) cholesterol (C) levels. Study participants were men and women 19 to 80 years of age with baseline plasma low density lipoprotein (LDL) C levels > or = 4.5 mmol/l (175 mg/dl), HDL-C levels < or = 1.2 mmol/l (45 mg/dl), and triglyceride levels < or = 3.4 mmol/L (300 mg/dl). All subjects were stabilized on a diet for eight weeks prior to entry into two different protocols. In the first protocol 229 subjects were randomized to placebo or extended-release gemfibrozil (1200 mg/day) for 3 months (placebo trial). In the second protocol 655 subjects were randomized to regular or extended-release gemfibrozil (1200 mg/day) for 6 months (equivalency trial). Changes in lipids and apos were stratified by baseline HDL-C levels (< 0.9 mmol/l, and 0.9-12.2 mmol/l). In both studies, treatment with gemfibrozil, either regular or extended-release, was associated with significant (P < 0.05) decreases in plasma very low density lipoprotein (VLDL) C and triglyceride levels of 42-45% and 33-37%, respectively, in subjects with HDL-C level < 0.9 mmol/l, and of 38-47% and 32-39%, respectively, in patients with HDL-C levels of 0.9-1.2 mmol/l. Modest reductions from baseline in directly measured LDL-C levels were observed in both groups (3-6% and 8-9%, respectively). These reductions were less than those observed for calculated LDL-C (7-10% and 11%, respectively). For apo B, reductions were 11-14% and 16-17% in the two groups. HDL-C, apo A-I, and apo A-II levels increased by 15-16%, 5-6%, and 21-25%, respectively, in patients with HDL-C < 0.9 mmol/l, and by 6-7%, 2-3%, and 19-22%, respectively, in patients with HDL-C of 0.9-1.2 mmol/l. These differences in HDL-C levels reached statistical significance in the equivalency trial (P < 0.0001) and were independent of baseline triglyceride levels. Our data indicate that gemfibrozil, either regular or extended-release, is highly effective in lowering plasma triglyceride levels and increases HDL-C levels by approximately 15% in hypercholesterolemic patients with low HDL-C levels (< 0.9 mmol/l). Moreover, this agent lowers VLDL-C somewhat more than triglyceride, resulting in an underestimation of calculated VLDL-C reductions and in an overestimation of calculated LDL-C reductions. This agent also raises apo A-II levels much more than apo A-I levels.     

Plasma lipoprotein distribution of apoC-III in normolipidemic and hypertriglyceridemic subjects: comparison of the apoC-III to apoE ratio in different lipoprotein fractions

In order to assess the relationship between plasma accumulation of triglyceride-rich lipoproteins (TRL) and lipoprotein levels of apoC-III and apoE, we have measured apoC-III and apoE in lipoproteins separated according to size (by automated gel filtration chromatography) from plasma of normolipidemic subjects (plasma triglyceride (TG): 0.84 +/- 0.10 mmol/l; mean +/- SE, n = 8), and from type III (n = 8) and type IV (n = 8) hyperlipoproteinemic patients, matched for plasma TG (5.76 +/- 0.62 v 5.55 +/- 0.45 mmol/l, resp.). Total plasma apoC-III concentration was similar in type III and type IV patients (33.1 +/- 3.4 v 37.6 +/- 4.4 mg/dl, respectively), but was significantly increased compared to normolipidemic controls (10.0 +/- 1.0 mg/dl, P < 0.001). TRL apoC-III was lower and high density lipoprotein (HDL) apoC-III was significantly higher in type III versus type IV subjects (14.8 +/- 3.2 vs. 22.8 +/- 3.0 mg/dl, P < 0.05; 8.3 +/- 1.0 vs. 5.2 +/- 0.5 mg/dl, P < 0.05). Plasma concentration of apoC-III in lipoproteins that eluted between TRL and HDL (intermediate-sized lipoproteins, ISL) was similar in the two hypertriglyceridemic groups (10.1 +/- 1.3 vs. 9.7 +/- 1.6 mg/dl), but was significantly higher (P< 0.05) than controls (2.2 +/- 0.3 mg/dl). TRL, ISL, and HDL apoE concentrations were significantly higher in type III versus type IV subjects (P < 0.05). All lipoprotein fractions in type III patients were characterized by lower apoC-III to apoE ratios. In contrast, the TRL apoC-III to apoE ratio of type IV patients was similar and the ISL apoC-III to apoE ratio was significantly higher, compared to normolipidemic individuals. These results indicate that compared to normolipidemic individuals, remnant-like lipoproteins in the ISL fraction of type IV patients are enriched in apoC-III relative to apoE, whereas those of type III patients are enriched in apoE relative to apoC-III.     

Renal and metabolic effects of 1-year treatment with ramipril or atenolol in NIDDM patients with microalbuminuria

The clinical importance of selection of different antihypertensive drugs for the treatment of diabetic patients is still unclear. Thus we performed a randomised, controlled study in 105 hypertensive non-insulin-dependent diabetic (NIDDM) patients with microalbuminuria over 1 year. Patients received either the angiotensin converting enzyme (ACE) inhibitor ramipril (2.5-5.0 mg/day; in addition 24% of patients also received felodipine) or the beta blocking agent atenolol (50-100 mg/day; in addition 24% of patients also received hydrochlorothiazide). Blood pressure, metabolic control, lipid levels and albumin excretion rate were studied during the follow-up. After 1 year an almost identical fall (p < 0.001) in blood pressure was observed with ramipril (170/100 vs 150/ 85 mmHg, median) and atenolol (180/100 vs 150/ 80 mmHg, median). With ramipril a reduction of total cholesterol (6.3 vs 5.9 mmol/l), of LDL cholesterol (3.8 vs 3.6 mmol/l) and HDL cholesterol (1.3 vs 1.2 mmol/l) was found, whereas triglycerides slightly increased (1.8 vs 2.0 mmol/l). With atenolol a similar reduction of total cholesterol (6.3 vs 5.9 mmol/l), LDL cholesterol (3.8 vs 3.7 mmol/l) and HDL cholesterol (1.4 vs 1.2 mmol/l) and an increase of triglycerides (1.4 vs 1.7 mmol/l) was noted. Metabolic control of the patients was maintained with both ramipril and atenolol treatment. With ramipril treatment urinary albumin creatinine ratio (14.4 vs 13.8 mg/mmol) and creatinine clearance (82 vs 84 ml/min) were constant, but with atenolol an increase of albumin creatinine ratio (13.9 vs 19 mg/mmol, p < 0.001) and a slight decrease of creatinine clearance (80 vs 66 ml/min, p < 0.05, not significant after Bonferroni correction) was observed. In conclusion: 1-year treatment of NIDDM patients with ramipril or atenolol does not influence metabolic control, the changes in serum lipids were similar. Despite almost identical blood pressure reduction in both groups the albumin creatinine ratio was constant under ramipril, but increased under atenolol treatment.     

Association of Helicobacter pylori infection with elevated serum lipids

Helicobacter pylori causes a chronic gastric infection, which has been associated with coronary heart disease. To evaluate the mechanisms of this association, we studied whether the infection affects serum lipid levels as previously shown in acute infections. We analysed the serum samples of 880 males who participated in a reindeer herders' health survey in Northern Finland in 1989. H. pylori IgG and IgA antibodies were measured by enzyme-linked immunosorbent assay and triglyceride, total cholesterol and high-density lipoprotein cholesterol concentrations by routine enzymatic methods. A total of 52% of the subjects were positive for both H. pylori specific IgG and IgA and 31% were antibody-negative. The serum triglyceride and total cholesterol concentrations were significantly higher in the males with positive IgG and IgA antibody titres for H. pylori than in the males with no signs of infection (1.20 vs. 1.03 mmol/l, P < 0.001 and 6.59 vs. 6.11 mmol/l, P < 0.001, respectively). The associations remained statistically significant in non-smokers after the adjustment for age, body mass index (BMI) and social class. The finding supports the hypothesis that chronic infections may modify the serum lipid profile in a way that increases the risk of atherosclerosis.     

Magnesium status in healthy free-living elderly Norwegians

With evidence on the important role of glycosaminoglycans (GAG) in the pathogenesis of endocrine ophthalmopathy (EO) having accumulated, the present study focused on the biochemical assessment of plasma GAG content in 37 EO patients as compared to 20 controls. Glycosaminoglycans were isolated from plasma samples by protein elimination, dialysis, and precipitation with ethanol and cetylpyridinium chloride. Patients (9.71, 5.09, 15.09 mg/100 ml; median, 25th, 75th percentile) exhibited significantly (p = 0.0021) higher plasma GAG levels than controls (4.6, 3.38, 6.8 mg/100 ml). Plasma GAG content was unrelated to age, sex, or antithyroid treatment. However, an even higher level of significance (p = 0.0001) was reached when discriminating between untreated patients with EO of recent onset (14.16, 10.35, 15.51 mg/100 ml) and controls. By contrast, steroid therapy of EO led to values (3.82, 1.85, 6.52 mg/100 ml) indistinguishable from those of the controls. Further statistical analysis of the results, based on a specificity of 95% for the control group, revealed a sensitivity of 91% for patients with untreated EO of recent onset, and a specificity of 100% for patients receiving steroid therapy. In comparison, plasma GAG content was determined in 8 untreated and in 6 treated EO patients by a second method already published. All untreated patients exhibited high GAG levels (median 2.23 mg/100 ml) whereas in treated EO patients normal plasma GAG values (0.17-0.34 mg/100 ml) were found. Follow-up determination of plasma GAG content in 7 patients undergoing steroid treatment unveiled a marked decrease of initially elevated values. These findings correlated well with clinical improvement of thyroid eye disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and sensitive determination of catecholamines and the metabolite 3-methoxy-4-hydroxyphen-ethyleneglycol using HPLC following novel extraction procedures

In the present study assays were improved for the determination of free catecholamines and 3-methoxy-4-hydroxyphenethyleneglycol (MHPG), the major metabolite of peripheral and central noradrenaline. The compounds were extracted by a fluid phase extraction: a diphenyl boric acid method for the purification of catecholamines and an ethyl acetate extraction for MHPG were used, respectively. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. In previous studies, significant differences between plasma concentrations of these substances in normal volunteers and hospital patients were demonstrated. Therefore, we established valid reference values for a hospital population. Blood and urine samples of 59 patients without disorders and medication affecting catecholamine synthesis and metabolism or the activity of the sympatho-adrenal system were collected and analyzed for free and total (free plus conjugated) MHPG, noradrenaline (NA), adrenaline (A) and dopamine (DA); total MHPG was assayed after enzymatic hydrolysis of conjugates. Our data clearly demonstrate that these methods are sensitive, specific, rapid, and can easily be standardized. The intra- and inter-assay precision were high (CV 2.6-5.3% and 4.3-6.9% for plasma, CV 3.8-4.9% and 5.1-8.2% for urine, respectively). For plasma, the mean concentrations +/- SD were determined to be 20.82+/-4.70 pmol/ml for free MHPG, 68.43+/-16.21 pmol/ml for total MHPG, 2.11+/-0.24 pmol/ml for NA and 0.31+/-0.08 pmol/ml for A. For 24h-urine the mean concentrations +/-SD were determined to be 0.44+/-0.13 mmol/24h for free MHPG, 8.79+/-2.13 mmol/24h for total MHPG, 169.8+/-58.25 nmol/24h for NA, 62.19+/-21.79 nmol/24h for A and 757.2+/-382.6 nmol/24h for DA. In summary, these novel and rapid methods can clearly be employed in a routine clinical setting.     

Pathway engineering for the production of aromatic compounds in Escherichia coli

The standard enzymatic assay for quantification of D-sorbitol in plasma was adapted to the automatic analyzer Cobas Mira S. In the assay, NAD (reagent) in the presence of sorbitoldehydrogenase (SDH; start reagent) converts D-sorbitol to fructose with formation of NADH, which was detected automatically as the difference between the first and last readings at 340 nm. The sample blank values for each specimen were subtracted to exclude both endogenous D-sorbitol and sugars, which also react as substrates for SDH. The method is simple, rapid (40 samples/h), precise down to endogenous concentrations (coefficient of variation < 5%; limit of determination: 0.38 mg/L) and linear up to 100 mg/L. Samples with higher D-sorbitol concentrations were estimated after dilution. The method was used to measure disposition curves of sorbitol in volunteers after a single intravenous dose of 0.8 g sorbitol.     

Determination of flurbiprofen and ibuprofen in dog serum with automated sample preparation

Methods for the determination of flurbiprofen and ibuprofen in dog serum were developed using high-performance liquid chromatography and automated serum extraction. Sample extraction was automated by use of cartridges packed with a styrene-divinylbenzene macroreticular resin in a microprocessor-controlled centrifugal system. The average recoveries were 98.9% for flurbiprofen and 94.5% for ibuprofen. The limits of detection were approximately 0.04 microgram/ml for flurbiprofen at 254 nm and 0.5 microgram/ml for ibuprofen at 230 nm. The relative standard deviations for the determination of a laboratory standard between days was 2.4% (20 microgram/ml) for flurbiprofen and 1.7% (13 microgram/ml) for ibuprofen. Peak height ratios were linear with concentrations of 0.04--100 microgram/ml for flurbiprofen and 1.0-50 microgram/ml for ibuprofen. These methods are simple, rapid, sensitive, and specific. The use of an automated sample preparation procedure improved the between-day precision by a factor of two when compared to a manual extraction procedure. These methods were applied to bioavailability studies in dogs.