Statistical approach for evolutionary relationship of alpha-conotoxins and members of insulin-superfamily.

Similar conserved structures appear in apparently unrelated protein families. Thus, the superfamily of insulin shows an evolutionary relationship with the alpha-conotoxins of marine fish-hunting snails as indicated by methods of protein comparison. In order to reach statistical significance, the A-chains of different insulins, insulin-like growth factors, relaxins, insulin related peptides from invertebrates were drawn for comparison. These data were correlated with sequences from randomly chosen proteins. The alpha-conotoxins show identity scores up to 37.5% and similarity up to 56.2% toward the members of the insulin-superfamily. These scores conform to values achieved by comparing the relaxin and the insulin/IGF-sequences. The data show clearly that the identity and similarity values obtained in the comparison with the insulins are significantly higher than the scores of randomly chosen protein primary structures. According to our calculated data, this hormone system regulating metabolism and growth in vertebrates and the mentioned toxin-receptor system share the same evolutionary ancestor. However, this statistical approach has to be substantiated on gene level.

Subcutaneous glucose concentration in humans. Real estimation and continuous monitoring.

OBJECTIVE: To determine the real subcutaneous glucose concentration in healthy volunteers to help in the development of new calibration methods for subcutaneous glucosensors. RESEARCH DESIGN AND METHODS: We developed a new method to estimate the real subcutaneous glucose concentration based on the recirculation of phosphate-buffered saline (PBS) in a microdialysis probe inserted into the subcutaneous tissue. Tissue glucose diffuses into the probe until complete equilibration between the glucose concentration outside and inside the microdialysis probe is achieved. Later, the glucose content of the recirculated PBS is assessed in vitro. We applied the method in 10 healthy volunteers under fasting state and during a hyperglycemic clamp. In addition, we monitored the subcutaneous glucose with an enzymatic-amperometric glucosensor combined with a microdialysis probe. RESULTS: The subcutaneous glucose concentration measured by the recirculation method was 72 +/- 6 and 78 +/- 6% of the blood glucose measured in the fasting state and during the hyperglycemic clamps, respectively. On the other hand, the glucosensor's signal correlated significantly with the blood glucose. CONCLUSION: The recirculation method estimated the real subcutaneous glucose concentration, opening the way to develop new calibration procedures for subcutaneous glucosensors. However, a suitable calibration procedure is still lacking.

Quality control of intensified insulin therapy: HbA1 versus blood glucose.

Since the measurement of HbA1 has become available to the diabetologists, the physicians and patients tend to rely exclusively on this parameter for the assessment of metabolic control. Therefore, in this study, 24 hour glucose profiles of 8 selected patients (4 under intensified conventional therapy, ICT, and 4 under continuous subcutaneous insulin infusion, CSII) with HbA1 values indicating good metabolic control were taken three times at four week intervals and were compared to mean blood glucose (MBG), mean average of glucose excursions (MAGE) and Schlichtkrull's M-value. MBG of the 24 profiles was 114 +/- 21 mg/dl. The patients under CSII were somewhat lower than the patients under ICT. In 16 of the 24 profiles, there was at least one period of hypoglycemia of 50 mg/dl and below. Only in one patient, M-value and MAGE showed stable metabolic control. In conclusion, hyperglycemic excursions in patients under intensified conventional therapy or treated by continuous subcutaneous insulin infusion do escape their reflection in the HbA1 values because of low blood sugar periods following hyperglycemic swings. Undoubtedly, the partial failure of ICT and CSII to prevent diabetic complications might be ascribed to the incomplete assessment of the metabolic control based on HbA1 values exclusively.

the "Ulm Zucker Uhr System" and its consequences.

The "Ulm Zucker Uhr System" comprises a microdialysis probe, a biosensor (Glucosensor Unitec Ulm s.c.), a sender transferring telemetrically the glucose concentrations, and a receiving indicator. The "Zucker Uhr" or "Sugar Watch" looks and operates like a normal wrist watch. We now present the functioning pre-prototype of the first portable system, which 1) is continuously measuring in s.c. tissue the Tissue Glucose (T.G.) concentration in the intercellular fluid on-line enzymatically by combining an enzymatic and an electrochemical (amperometric) technique. 2) is transferring in the patient via the "Zucker Uhr" once per minute the actual tissue glucose concentration, i.e. 1,440 values/24 hours or even 2880 values/48 hours, 3) is actually alarming the patient by optical and acoustic means, when the tissue glucose is too high (hyperglycemia) or too low (hypoglycemia), 4) is storing digitally all values and, therefore, permitting the dialogue between doctor and patient as to his diabetes control in view of his 24 h glucose profile, which is visual on a computer screen. The advantage for the patient is on the one hand the elimination of the multiple pricking in the finger tips for providing capillary blood, and on the other hand, permitting the preview of the limitation and perhaps the complete elimination of the microangiopathy of the retina, kidneys and nervous system. The trend of the T.G. decreases is readily recognizable, which indicates immediately the dangerous hypoglycemia. Likewise, over 48 hours, the hyperglycemic periods are transmitted and recorded which are not measured and reflected by the HbA1 and HbA1c conventional measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Calibration problems of subcutaneous glucosensors when applied "in-situ" in man.

Continuous glucose monitoring is the conditio sine qua non to achieve total automation in glucose-controlled insulin-delivery. Several types of glucosensors have been designed according to the enzyme-amperometric method to measure the glucose in different human compartments. However, problems such as long-term stability and calibration prevent this technique being put into practice. A feasible method is needed to calibrate the glucosensor and at the same time should be accepted by the patients. To achieve calibration we determined the absolute tissue glucose, as well as the microdialysis recovery in-vivo, in healthy subjects under normal conditions and during a hyperglycaemic clamp by applying a device based on the recirculation of phosphate buffer saline in a microdialysis probe implanted in the s.c. adipose tissue. The first experiments carried out were promising and encouraging, but further investigations are still needed to favour an ideal "before implantation, all in-vitro" method to calibrate a s.c. glucosensor.

Combination of microdialysis and glucose sensor for continuous on line measurement of the subcutaneous glucose concentration: theory and practical application.

The microdialysis technique can be used to get dialysates of the subcutaneous tissue, which can be continuously measured by an amperometric glucose sensor. In order to get further insight into the microdialysis procedure, we used a steady-state theory for microdialysis to predict the recovery of glucose in the dialysate and compared the results to experimental data obtained by a combination of the microdialysis technique with continuous amperometric glucose sensing. The recovery of glucose obtained in vitro for two different microdialysis probes was close to the theoretical predictions. When quantifying the predictions of the model with regard to the spatial concentration profile in the subcutaneous tissue, it appeared, that the presence of the microdialysis probe depressed the concentration of glucose for 0.2 mm from the probe surface. In a 24 hour in vivo experiment, there were less fluctuations in the sensor signal when the patient was lying in bed compared to the time, when the patient could move freely. In conclusion, the combination of microdialysis and glucose sensor seems to be a promising approach to a continuously functioning glucose sensing system. However, the microdialysis procedure itself disturbs the surrounding of the probe leading to a concentration gradient of glucose. This might explain some differences between the course of blood glucose and the course of subcutaneous glucose, measured by the combination of microdialysis and an amperometric glucose sensor. Further developments of such systems should aim at implanting microdialysis devices which have a minimal influence upon the tissue metabolism.

Long-term experiences with a computerized diabetes management and glucose monitoring system in insulin-dependent diabetic patients.

Only a minority of patients use diabetes management systems. In our study, 24 patients were given a memory based blood glucometer (ROMEO, Diva Medical Systems), which additionally allowed the input of insulin doses, food intake and exercise for up to four years. Eight patients returned ROMEO within one month (group I), 8 used the system as long it was available as a loan (group II) and 8 patients bought the system (group III). The patients of group III had a significantly better diabetic control (HbA1 = 7.8% +/- 0.7 (S.D.) vs. 11.7 +/- 3.6 (group I) and 10.7 +/- 2.6 (group II)) and were more reliable in their input of the data. After 3 years, however, only five patients of group III continued to use the system, but as a glucose meter only. One patient used the options of the system. These data show that glycemic control is not a question of the equipment. After a certain period, even the well-motivated patients do not use the system options routinely. Obviously, the advantages of the system are only used by a minority of patients characterized by good metabolic control and good compliance, and even these patients do not persistingly use all the options of the system after a certain period of time.

Use of the microdialysis technique in the monitoring of subcutaneous tissue glucose concentration.

For some time the subcutaneous (s.c.) tissue has been the target for continuous glucose measurement. The microdialysis technique permits an extracellular region approach, which has been used for about two decades for measuring various metabolites in dialysates obtained from different body regions. By connecting a s.c. implanted microdialysis probe to a flow chamber of an amperometric glucose sensor, the procedure of glucose sensing was transferred to ex vivo. Using this device it was possible to obtain, for up to 24 hours, s.c. tissue glucose profiles of healthy and diabetic people. The microdialysis theory, the calibration process and other microdialysis technique applications are discussed in this paper. Although the combination of the microdialysis technique and amperometric glucose sensing requires certain technical equipment, the combination of microdialysis and glucose sensor seems to be a promising approach to a continuously functioning glucose sensing system.

On line continuous monitoring of subcutaneous tissue glucose is feasible by combining portable glucosensor with microdialysis.

A new method for continuous measurement of subcutaneous tissue glucose content is introduced: by combining the microdialysis technique with a wearable amperometric glucose sensor, a device for continuous glucose measurement in the subcutaneous tissue was obtained. This device was applied to healthy volunteers (n = 10) over the period of an oral glucose load and to type I diabetic patients (n = 10) under the conditions of daily life. Glucose profiles in both healthy and diabetic persons were followed in the subcutaneous tissue up to 27 hours. This technique will certainly open new perspectives of monitoring and treating diabetic patients.

On line continuous monitoring of blood lactate in men by a wearable device based upon an enzymatic amperometric lactate sensor.

A wearable device for the continuous measurement of lactate in the blood was constructed by the combination of continuous blood sampling employing a double lumen catheter with an amperometric lactate sensor. In vitro, the lactate sensor turned out to have a linear concentration range between 0 and 15 mmol/l. The response time of the sensor itself amounted to 100 sec, whereas the lag time for blood sampling amounted to 2.2 min. In vivo, the lactate sensor was successfully used for the detection of changes of the blood lactate concentration following strenuous exercise in 7 healthy volunteers, in two cases up to 22 h. In conclusion, the technique of continuous blood sampling by the use of a double lumen catheter in combination with the amperometric lactate sensor is feasible and simplifies frequent blood lactate estimations.

The function of a hydrogen peroxide-detecting electroenzymatic glucose electrode is markedly impaired in human sub-cutaneous tissue and plasma.

Electroenzymatic glucose sensors implanted into sub-cutaneous (s.c.) tissue of human subjects and experimental animals exhibit lower sensitivities to glucose than in buffer solutions before implantation. The mechanism of the decrease of sensitivity is not known. Sensors used in this study were fabricated from platinum wires (diameter 0.125 mm) with covalently bound glucose oxidase at the tip of the wire. After coating the tip with polyurethane, wires were placed into 27 gauge steel needles. Sensors were operated potentiostatically at 700 mV against Ag/AgCl pseudo-reference electrodes. These sensors were implanted s.c. in 6 diabetic patients for 7 h. In 4 patients, sensors were responsive to successive increases of plasma glucose levels. Mean sensitivity to glucose in s.c. tissue was 29% of in vitro sensitivity. In 2 patients there was a sudden decrease of sensor currents, unrelated to glucose, shortly after implantation. Sensors were inhibited in human plasma to a similar extent. When sensors were exposed to native plasma and to plasma ultrafiltrate (mol. wt. < 10 kDa) for 10 h, identical decreases of signals were found. Exposure to dialysed plasma (mol. wt. > 12 kDa) caused much less decrease of sensor signals. Losses of sensor sensitivities to glucose in s.c. tissue and in plasma were totally reversible upon re-exposure of sensors to buffer solutions. We conclude that sensor inactivation in plasma and possibly in s.c. tissue is caused by low molecular weight substances not retained by the polyurethane membrane.

Fluctuations of carbohydrates in haemolymph of honeybee (Apis mellifica) after fasting, feeding and stress.

The haemolymph of honeybee contains trehalose, a non-reducing disaccharide, glucose and fructose. The aim of this work was to describe changes of haemolymph sugar induced by different physiological situations. Gas chromatography for the determination of carbohydrate concentrations in the haemolymph was introduced. The individual bee was anesthetized by CO2 and punctured at tergum III in order to gain 1 microliter haemolymph using a glass capilette. The main sugars in the haemolymph were determined as silylated derivatives by gas chromatography. Bees living under exactly standardized conditions in an artificial bee-house were observed after feeding and fasting. In addition saccharides were determined after "run-stress" along different distances.

On line continuous monitoring of subcutaneous tissue glucose in men by combining portable glucosensor with microdialysis.

For the normalisation of blood glucose levels in diabetic patients by feedback controlled insulin delivery, a self-manageable and reliable method for continuous glucose estimation is still not available. By combining a commercially available needle type dialysis probe (molecular cutoff 20,000 Da) with a sensitive glucose sensor, we obtained a device for continuous glucose measurement in dialysate. This device was tested in healthy volunteers during a 75-g oral glucose tolerance test and in Type 2 (non-insulin-dependent) diabetic patients. Venous glucose and subcutaneous sensor signal were followed for 300 min (ten healthy subjects), 21 h (three healthy subjects) or 9 h (seven Type 2 diabetic patients). The recovery of the microdialysis was interindividually different, but after calibration, glucose levels in the dialysate and subcutaneous glucose sensor signal correlated well (r = 0.84-0.95). Under the assumption of a physiologic and technical delay between intravenous and subcutaneous glucose, correlation coefficient between intravenous glucose and subcutaneous sensor signal ranged from 0.60 to 0.93. We conclude that changes in blood glucose could be monitored in the subcutaneous tissue by the microdialysis technique in a continuous on line manner.