Advanced glycation end-products and the progress of diabetic vascular complications.

Epidemiological studies have confirmed that hyperglycemia is the most important factor in the onset and progress of vascular complications, both in Type 1 and 2 diabetes mellitus. The formation of advanced glycation end-products (AGEs) correlates with glycemic control. The AGE hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications including nephropathy, retinopathy, neuropathy and atherosclerosis. Recent studies have shown that increased formation of serum AGEs exists in diabetic children and adolescents with or without vascular complications. Furthermore, the presence of diabetic complications in children correlates with elevated serum AGEs. The level of serum AGEs could be considered as a marker of later developments of vascular complications in children with Type 1 and 2 diabetes mellitus. The careful metabolic monitoring of young diabetics together with monitoring of serum AGEs can provide useful information about impending AGE-related diabetic complications. It is becoming clear that anti-AGE strategies may play an important role in the treatment of young and older diabetic patients. Several potential drug candidates such as AGE inhibitors have been reported recently.

[Actions of standardized extracts of Crataegus berries on exercise tolerance and quality of life in patients with congestive heart failure]. / Wirksamkeit eines standardisierten Extraktes aus frischen Crataegus-Beeren auf Belastungstoleranz und Lebensqualität bei Patienten mit Herzinsuffizienz (NYHA II).

Standardized extracts of Crataegus leaves and blossoms are said to have positive inotropic, positive dromotropic and negative bathmotropic effects. Clinical trials produce evidence for an improvement of symptoms in patients with congestive heart failure (NYHA II). In this trial the efficacy of a standardized extract of fresh Crataegus berries (Rob 10) on exercise tolerance and quality of life was studied in 88 patients. In a three-month placebo-controlled, randomized, double-blind trial these patients were treated with Rob 10 (3 x 25 drops daily). Total exercise time in bicycle ergometry was defined as primary efficacy variable, while quality of life (Minnesota Questionnaire), Dyspnea-Fatigue Index and the assessment of dyspnea by the patient on a visual analogous scale were chosen as secondary parameters. Investigations were performed after a two week placebo run-in period as well as 6 and 12 weeks after the onset of the study. Treatment with Rob 10 led to a increase of exercise time of 38.9 s vs placebo (95% confidence interval 5.7-72.1 s). Quality of life improved accordingly in favour of Rob 10. In the Minnesota Questionnaire, the total score fell by 31% (30.6 vs 44.1) under Rob 10 vs 18% (34.6 vs 42.4) under placebo. The Dyspnea-Fatigue Index demonstrated an increase of the total score of 12% (9.41 vs 8.37) vs 8% (8.92 vs 8.26) under administration of placebo. According to findings of the assessment of dyspnea by the patient, dyspnea decreased by 11% (50.5 vs 56.6 mm) vs 4% (54.8 vs 57.3 mm) under placebo. The present study proves the efficacy and safety of a standardized extract of fresh Crataegus berries (Rob 10) in patients with congestive heart failure (NYHA II) regarding the parameters evaluated.

Effect of aminoguanidine and copper(II) ions on the formation of advanced glycosylation end products. In vitro study on human serum albumin.

Nonenzymatic glycosylation of proteins is a weighty consequence of hyperglycaemia in diabetes. This study examines the possible effect of copper(II) ions on the glycosylation of human serum albumin (HSA) and the resulting formation of advanced glycosylation end products (AGEs). HSA in phosphate-buffered saline was incubated with 100 mmol/l glucose. The effect of addition of copper(II) ions and/or aminoguanidine bicarbonate (CAS 2582-30-1) was investigated. The determination of AGE levels derived from glycosylated/glycoxidated HSA was performed using a specific spectrofluorometric assay (excitation 346 nm; emission 418 nm). The results showed that the addition of copper(II) ions to the incubation medium containing glucose increased the formation of AGEs. Further, in the presence of copper(II) ions, a significant blockade of aminoguanidine inhibitory effect on the formation of AGEs was observed.

Inhibition of nonenzymatic protein glycation and lipid peroxidation by drugs with antioxidant activity.

We studied the effects of aminoguanidine (AG), beta-resorcylidene aminoguanidine (RAG), DL-penicillamine (PNCA) and captopril on early and advanced glycation of human serum albumin (HSA). We also assessed inhibition of lipid peroxidation by AG and RAG in erythrocytes. Incubation of HSA with D-glucose (20 mM, 37 degrees C for 21 days) led to the formation of Amadori products and fluorescent advanced glycation end-products (AGE). Only PNCA markedly reduced the formation of Amadori products, while all tested compounds markedly reduced the formation of AGE. AG and RAG also inhibited malondialdehyde formation in erythrocytes incubated with hydrogen peroxide. Addition of AG at concentrations from 1 microM to 1 mM caused a 10-80% inhibition of lipid peroxidation. Thus, AG and RAG inhibit toxic oxidative processes and may have therapeutic potential in a number of human diseases.

Influence of the angiotensin II antagonist valsartan on left ventricular hypertrophy in patients with essential hypertension.

BACKGROUND: Left ventricular hypertrophy (LVH) represents an independent risk factor in patients with essential hypertension. Because reversal of LVH may be associated with an improvement of prognosis, the influence of new antihypertensive compounds, such as angiotensin II AT1 receptor antagonists, on LVH should be determined. METHODS AND RESULTS: In a randomized, double-blind trial, 69 predominantly previously untreated hypertensive patients with echocardiographically proven LVH, ie, left ventricular mass index (LVMI) >134 g/m2 in men and >110 g/m2 in women and/or end-diastolic septal thickness >12 mm, received either the angiotensin II antagonist valsartan or atenolol for 8 months. Echocardiographic data of 58 patients were available. After 8 months of valsartan treatment (n=29), LVMI decreased from 127+/-23 to 106+/-25 g/m2 (ratio [R]=0.83; 95% CI, 0.79 to 0.87; P<0.0001 versus baseline). Under atenolol (n=29), LVMI decreased to a smaller extent, from 127+/-25 to 117+/-27 g/m2 (R=0.92; 95% CI, 0.86 to 0.98; P=0.0082 versus baseline). The mean reduction of LVMI came to 21 g/m2 under valsartan and only to 10 g/m2 under atenolol (R=0.91; 90% CI, 0.85 to 0.97 versus atenolol). Baseline mean blood pressure values were determined to be 163+/-12/101+/-6 mm Hg before treatment with valsartan and 160+/-14/103+/-6 mm Hg before atenolol treatment. After 8 months of treatment, mean blood pressure decreased to 146+/-13/90+/-7 mm Hg with valsartan and to 147+/-18/90+/-7 mm Hg with atenolol. Nine patients in the valsartan group and 8 patients in the atenolol group required additional medication with hydrochlorothiazide. CONCLUSIONS: Antihypertensive treatment with the angiotensin II antagonist valsartan for 8 months produced a significant regression of LVH in predominantly previously untreated patients with essential hypertension. The drug may be safely administered in this subset of hypertensive patients; however, the long-term benefit in terms of risk reduction has still to be evaluated in further trials.

[The validity of radioimmunologic determination of bioavailability of beta-escin in horse chestnut extracts]. / Zur Validität radioimmunologisch bestimmter Bioverfügbarkeitsdaten von beta-Aescin in Rosskastaniensamenextrakten.

The bioavailability under steady state conditions of a standard, slow-release horse chestnut seed extract (HCSE)-containing product was compared with that of an analogous, fast-release test preparation (Noricaven novo) in a prospective, randomised, double-blind study in a double cross-over design. The serum concentration of beta-escin (CAS 6805-41-0) was measured by radioimmunoassay. In addition, the biopharmaceutical properties of the HCSEs present in the products were investigated, the amount and composition of the active ingredient, escin, being analysed with a validated HPLC method. The pharmacokinetics of this study were compared with the corresponding data of a similar investigation carried out under analogous conditions concerning study design, analytical methods and reference preparation. Comparison of the similar studies revealed differences in characteristic pharmakokinetic values of beta-escin in terms of a shift of the concentration time curves as could be demonstrated for the reference product. The total amounts of escin in the two products investigated did not differ significantly. However, quantitative and qualitative differences were detected in the constituents of the two different extract preparations. It is concluded that the high specificity of the validated beta-escin radioimmunoassay leads to analytical imprecision due to the variable constituents of the extract preparations used. It is necessary to test whether this problem can be solved using an analytical approach, which is specific for each extract.

Influence of isradipine and spirapril on left ventricular hypertrophy and resistance arteries.

Left ventricular hypertrophy is a common clinical feature in hypertensive patients and may be associated with structural changes in vessel morphology. In an open prospective trial, we evaluated 14 patients with previously untreated hypertension (163 +/- 2/104 +/- 2 mm Hg) and an echocardiographically determined left ventricular mass index of 141.6 +/- 5.2 g/m2, indicating left ventricular hypertrophy. We obtained a gluteal skin biopsy sample before starting treatment to investigate subcutaneous small-artery (approximately 200 to 400 microns diameter) morphology and function. Patients then received antihypertensive treatment with a combination of spirapril (3 or 6 mg) and isradipine (2.5 or 5 mg). Echocardiographic recordings were made after 6 months and 1 year, and a final biopsy was taken after 1 year. After 1 year, blood pressure was significantly reduced to 142 +/- 3/ 90 +/- 1 mm Hg (P < .001), and left ventricular mass index decreased significantly to 105.3 +/- 5.8 g/m2 (P < .001). Baseline media-lumen ratio (7.64 +/- 0.48%) was not markedly reduced (7.21 +/- 0.55%), although a decrease occurred in 7 of 12 evaluable patients. Norepinephrine-induced vasoconstriction was markedly reduced after 1 year. In conclusion, a significant regression of left ventricular hypertrophy was obtained after 1 year of treatment with spirapril and isradipine, whereas a similar reduction in medial thickness relative to lumen diameter of subcutaneous small arteries could not be observed in all patients. Reversal of structural changes in resistance vessels may require a longer treatment period in patients with proven left ventricular hypertrophy.

HPLC analysis of azathioprine metabolites in red blood cells, plasma and urine in renal transplant recipients.

Anemia has been frequently reported in renal transplant recipients receiving azathioprine for immunosuppression and enalapril for treatment of hypertension. During the course of a prospective trial in such patients we determined azathioprine metabolites in erythrocytes, plasma, and urine as well as erythropoietin and hemoglobin levels in order to evaluate a potential interaction between these 2 drugs, possibly leading to anemia. Two specific high performance liquid chromatography (HPLC) methods for determination of azathioprine metabolites, both employing a mercurial cellulose resin for extraction, are presented. One method using a strong anion exchange column allows detection of 6-thioguanosine di- and triphosphate (thioguanine nucleotides) in red blood cells (RBC) with a sensitivity of 30 pmol/100 microliters RBC. 6-mercaptopurine (MP) and 6-thiouric acid (TUA) in plasma and urine were analyzed simultaneously by reversed-phase HPLC with a sensitivity of 5 ng/ml. The average (median values are given) steady state concentrations of thioguanine nucleotides in erythrocytes came to 267 pmol/100 microliters RBC (range 53-613) with and to 246 pmol/100 microliters RBC (range 39-629) without concomitant enalapril medication. Mean plasma concentrations of MP and TUA 3 hours after drug intake came to 14.8 +/- 9.9 ng/ml and 398 +/- 262 ng/ml, respectively, during enalapril comedication. Withdrawal of enalapril did not influence these metabolite levels coming to 15.3 +/- 9.1 and 451 +/- 253 after stopping enalapril treatment. Thioguanine nucleotides in RBCs were neither related to the dose of azathioprine given (r = -0.113, p > 0.05) nor to hemoglobin levels (r = 0.278, p > 0.05). However, azathioprine dose/kg body weight seemed to be related to hemoglobin concentration, with and without enalapril comedication. We conclude that enalapril therapy does not influence the measured azathioprine metabolites, the reported cases of anemia may rather be due to a pharmacodynamic interaction as shown by the significant increase in erythropoietin after withdrawal of enalapril. The assays described here are suitable to study the metabolism of azathioprine in patients with various diseases.

Oxygen release kinetics in healthy subjects and diabetic patients. II: Effects of HbCO.

Glycosylated hemoglobin (HbA1c) and carboxyhemoglobin (HbCO) are elevated in diabetic smokers. Both increase the oxygen affinity of hemoglobin and lower the velocity of oxygen release. We have, therefore, measured the influence of HbCO on the oxygen dissociation kinetics of hemoglobin in blood from healthy subjects (HbA1c = 5.3 +/- 0.3%, n = 12) and diabetic patients (HbA1c = 8.4 +/- 1.6%, n = 12) using the stopped flow technique. In addition, the effect of 2,3-DPG on the oxygen dissociation rate of hemoglobin containing high concentrations of HbCO was examined. Neither statistically nor clinically significant differences in the oxygen dissociation rate between blood from healthy subjects and diabetic patients were found. Increasing the HbCO concentration up to 20% of total hemoglobin produced an approximately 20% decrease (p < 0.001) in the dissociation rate constant in blood samples from both groups of subjects. Addition of 20 mmol 2,3-DPG per 10 mmol hemoglobin had little effect on the magnitude of these changes. It is concluded that HbCO values similar to those present in smokers cause a significant decrease of oxygen release in healthy subjects and diabetic patients. Elevated HbA1c concentrations do not potentiate the effects of HbCO on oxygen release in either group of subjects.

Oxygen release kinetics in healthy subjects and diabetic patients. I: The role of 2,3-diphosphoglycerate.

Glycosylated hemoglobin (HbA1c), found in higher concentration in diabetic patients (average 9% versus 5% in healthy subjects), has an approximately ten-fold higher oxygen affinity than non-glycosylated hemoglobin (HbA0). But the oxygen affinity in blood from diabetic patients does not differ from that in healthy subjects. Thus, it was concluded that 2,3-DPG, which lowers the oxygen affinity of hemoglobin by stabilizing deoxyhemoglobin and which is increased in diabetic patients, compensates for the effect of HbA1c. Therefore, oxygen release kinetics of hemoglobin in diabetic patients (HbA1c = 9.3 +/- 0.3%) and healthy subjects (HbA1c = 5.2 +/- 0.3%) were compared and the influence of 2,3-DPG determined using the stopped flow technique. The oxygen dissociation rate constant (k) of hemoglobin from the two groups did not differ (diabetic patients, 64.4 +/- 3.1 s-1 and healthy subjects, 65.1 +/- 2.3 s-1). Addition of supraphysiological concentrations of 2,3-DPG produced physiologically insignificant changes in this parameter. It is concluded that 2,3-DPG may not be the major factor compensating for the effect for HbA1c on oxygen release in diabetic patients.

Drug utilization review on a surgical intensive care unit.

Drug use reviews (DUR) provide information about drug prescription patterns. At the surgical intensive care unit (SICU) of the University Hospital Frankfurt/Main we recorded the indication related administration of blood products and pharmaceuticals (which were responsible for 60% of the total costs at the SICU in the year before) over a 4-month investigation period. Data were recorded and analyzed using a notebook-PC. 207 Patients were included, and recorded total expenditures came to $551,592. The 10 leading substances, represented by antibiotics and blood-products, caused more than 70% of total costs. The usage of these high-cost substances and the most expensive complications are identified and discussed. We concluded that DURs are useful for obtaining information about drug usage patterns and for identifying high cost drugs, which are of economic interest. Furthermore, the indications for the application of these substances can be identified. Our results suggest, that blood and blood-products should always be included in DURs. In our opinion, especially the usage of blood-products represents a field where considerable cost savings may be expected. As certain complications during the postoperative course (e.g. sepsis) may increase costs, they should be considered in the reimbursement negotiations between hospitals and health insurance.

Concentration/Effect Analysis of Antihypertensive Drugs.

Studies on the concentration/effect relationship of different antihypertensive agents (mainly calcium antagonists and alpha(1)-blockers) in patients confirm a close relation between plasma concentrations and the corresponding fall in blood pressure, but the heterogenity in study objectives and methodology impedes the compiling and differentiation of results. Concentration/effect analysis was established using either the linear or the E(max) model. The studies reviewed here do not speak unequivocally in favor of either one of the methodological approaches (linear or E(max) model, PK/PD modeling, placebo correction) or a certain mode of blood pressure measurement. The responsiveness parameters obtained exhibited a wide intersubject variability; thus, threshold levels derived for any of the investigated compounds can only be used to a limited extent. For drug development, a more integrated protocol should be strived for to design studies on concentration/effect relationship for antihypertensives. Although individualized dose adjustment based on concentration/effect analysis after single-dose application was applied successfully, this approach seems to be an inadequate strategy for conventional treatment of hypertension.

Effect of 2,3-diphosphoglycerate on O2-dissociation kinetics of hemoglobin and glycosylated hemoglobin using the stopped flow technique and an improved in vitro method for hemoglobin glycosylation.

Studies under equilibrium conditions have shown that the oxygen affinity of hemoglobin (Hb) is lowered by 2,3-diphosphoglycerate (2,3-DPG), the physiological allosteric effector in erythrocytes, and enhanced by glycosylation of Hb. The kinetics of oxygen release, as a function of 2,3-DPG and the degree of glycosylation have been determined using the stopped flow method and a new in vitro glycosylation procedure allowing adequate amounts of functionally intact hemoglobin to be obtained. The rate constant k of O2-dissociation in glycosylated Hb (8% HbA1c) was approximately 10% lower than in native Hb (4% HbA1c). The addition of 2,3-DPG in concentrations up to 20 mmol/l resulted in a progressive increase of k from 61.5 +/- 3.3 s-1 to 65.3 ae 4.1 s-1 for native Hb and from 56.8 +/- 5.2 s-1 to 59.4 +/- 4.1 s-1 for glycosylated Hb. We conclude that (a) the degree of glycosylation similar to that found in diabetic patients is responsible for a significant decrease of the oxygen dissociation velocity and (b) 2,3-DPG concentration similar to those occurring in vivo have only a weak effect on the oxygen dissociation velocity.