Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic.

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.

2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy.

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.

Fluoresceinyl-ethylenediamine-ouabain detects an acidic environment in the cardiac glycoside binding site of Na+/K+-ATPase.

To probe the pH value in the microenvironment of the cardiac glycoside-binding site of Na+/K+-ATPase, pH-sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino-ouabain (FEDO) had a pKs of 6.0 and showed a H+-dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na+/K+-ATPase at 37 degrees C and pH 7.25 in a slow time-dependent process under the conditions of backdoor phosphorylation with k(on) of 891 s(-1) M(-1). The complex dissociated with k(on) of 0.35 x 10(-3) s(-1) resulting in a Kd value of 0.4 microM for the FEDO x enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1 +/- 0.2 was calculated to exist in the microenvironment of the FEDO x enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO x enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO x enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern-Volmer constant from 6.2 mM(-1) (free FEDO) to 1.5 mM(-1) (FEDO x enzyme complex) indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO x enzyme complex by measurements of the anisotropy and the fluorescence half-life time revealed that both processes differed significantly when H2O was replaced by D2O. We conclude, therefore, that a pH of 5.1 +/- 0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain receptor complex has been formed.

DNA single-strand breaks in peripheral lymphocytes of clinical personnel with occupational exposure to volatile inhalational anesthetics.

The rates of DNA single-strand breaks in peripheral lymphocytes of 41 persons administering anesthesia daily and 44 control persons were determined by nucleoid sedimentation. There is a significantly higher rate of DNA single-strand breaks in nonsmoking anesthesia persons than in nonsmoking control persons (P < 0.01). Smoking anesthesia persons and smoking control persons presented increased rates of DNA single-strand breaks. Nonsmoking nurse anesthetists showed an insignificantly higher rate of damage than nonsmoking anesthesiologists. DNA single-strand breaks indicate damage before the start of DNA repair. Therefore, detected DNA single-strand breaks may be reversible. As not every DNA repair is perfect, increased rates of DNA single-strand breaks may possibly lead to irreversible DNA damage.

DNA single strand breaks in peripheral human lymphocytes after anesthesia with isoflurane-nitrous oxide-oxygen.

DNA single strand breaks were determined in peripheral lymphocytes of neurosurgical patients before and after 180 min of general anesthesia with isoflurane (CAS 26675-46-7)-nitrous oxide-oxygen. Immediately after anesthesia, the frequency of DNA single strand-breaks appeared to be significantly enhanced. In the majority of patients the DNA single strand breaks induced was equivalent to the effect of 0.2-0.5 Gray following x-ray radiation of lymphocytes in vitro. In a part of the examined patients these investigations were repeated on the first postoperative day. Then an increase of the frequency of DNA single strand breaks could not be demonstrated any more. The DNA single strand breaks were repaired by cellular repair systems. As DNA repair is regulated genetically, isoflurane-nitrous oxide-oxygen could induce DNA damage in patients with DNA repair defects.

DNA strand breaks in cells with DNA repair deficiency after halothane exposure in vitro.

Halothane (CAS 151-67-7) induced DNA strand breaks in isolated lymphocytes of two patients with a deficient DNA repair (xeroderma pigmentosum). In lymphocytes (resting cells) of healthy human donors and in L 5178 Y cells (proliferating cells) of mouse lymphoma, halothane did not induce demonstrable DNA strand breaks. The cells were exposed to 1.0 vol/% halothane for 60 min, and the DNA strand breaks were demonstrated by alkaline elution. The results suggest a possible genotoxic side effect of halothane in patients with deficiency in DNA repair.

Increasing DNase I activity after exposure of isolated DNA to halothane.

DNA was exposed to halothane (CAS 151-67-7) in a cell-free system. After exposure the DNA was used as substrate for DNase I from bovine pancreas. The DNase I activity increased after halothane exposure of the substrate depending on time and doses. Drugs are able to influence the DNA conformation. Conformational changes in the DNA can enhance the DNase I cleavage rate. Therefore, it is possible that halothane exposure induces changes in DNA conformation demonstrable by an increased DNase I activity. The results suggest a mechanism by which halothane may contribute to chromosomal defects and disturbances of DNA metabolism in cells.

[Micturition disorders following spinal anesthesia of different durations of action (lidocaine 2% versus bupivacaine 0.5%)]. / Miktionsstörungen nach Spinalanästhesie unterschiedlicher Wirkungsdauer (Lidocain 2% versus Bupivacain 0.5%).

Disturbances of micturition following spinal anaesthesia are considered to be rare and harmless side effects of this technique. For this reason, we set up a prospective study to investigate their incidence, characteristics and intensity. Our special interest was directed at the influence of the duration of action of local anaesthetics. METHODS. In a randomized, double-blind study, two groups, each consisting of 73 trauma surgical and orthopaedic patients, received isobaric spinal anaesthesia with either lidocaine 2% or bupivacaine 0.5%. From the 1st to the 3rd postoperative day, the patients were interviewed daily and asked specifically about disturbances of micturition. RESULTS. The two groups were comparable in terms of clinical data, spinal anaesthesia and surgery. Disturbances of micturition occurred only during the first 24 h and were observed in a total of 42%. They were about twice as frequent after bupivacaine (56%) as following lidocaine (27%). After bupivacaine there was a higher rate of difficult micturition or complete inability to micturate in the presence of an urge to urinate, carbachol medication and catheterization of the urinary bladder. Sex and age had no influence on the incidence. A history of disturbances of micturition increased their frequency. DISCUSSION AND CONCLUSIONS. Disturbances of micturition are the most common side effect of spinal anaesthesia during the first 24 h after surgery. Their higher frequency following the longer acting bupivacaine may be evidence of longer lasting blockade of the efferent sacral parasympathetic fibers innervating the detrusor vesicae muscle, leading to inhibition of bladder voiding. The consequences of these disturbances, if not correctly managed, may be distension of the urinary bladder with ensuing infection and loss of tone of the detrusor muscle. Various measures are recommended: choice of the longer acting local anaesthetic only if necessary, careful control of bladder filling, restrictive infusion of fluids, early mobilization, carbachol, catheterization in good time, prophylactic placement of an indwelling catheter in patients with previous disturbances.

[Sexuality following spinal anesthesia. A retrospective study of young men (less than 55 years old]. / Sexualität nach Spinalanästhesie. Eine Retrospektivbefragung bei jungen Männern (unter 55 Jahre).

A retrospective study was undertaken in order to determine whether the rumor that the use of spinal anesthesia in young men has a possible negative influence on sexual potency is justified or not. METHOD. Under the assumption that men up to the age of 55 years are generally sexually active, 1016 patients who were operated upon under spinal anesthesia (Ga. 22 and 25) during the last 5 years were requested to answer questions about their convalescence with emphasis on their pre- and postoperative sexual life. If they developed difficulties in this particular aspect, their opinion about the possible reasons was asked. RESULTS. Usable responses were obtained from almost 48% of the patients (Table 1). We excluded from the study those operations that could themselves have negatively influenced sexuality (Table 2). The patients belonged--as far as known--to ASA groups I-III (Fig. 2). Four-fifths of the patients had recovered completely 6 months after surgery (Fig. 4); their recovery was independent of the postoperative time interval (Table 1). Seventy-five percent were sexually active within 3 months after their operation (Fig. 6). There was no correlation between risk group and degree of recovery (Fig. 7, Tables 3 and 5). Nine of the preoperatively active patients did not have sex afterward, whereas 50% of the preoperatively inactive ones became active postoperatively (Tables 6 and 7). Four-fifths of the abstinent ones did not want sex or had no partner (Fig. 8). In no case was anesthesia mentioned as the cause of unwanted sexual abstinence (Fig. 9, Tables 3 and 4). Two patients who had resumed sexual activity but seemed dissatisfied with their sexual drive mentioned anesthesia as a possible cofactor in connection with surgery or psychological problems (Table 5). CONCLUSION. This is, as far as we know, the only study that has specifically investigated sexuality in relation to spinal anesthesia. The results show clearly that after uncomplicated, routine spinal anesthesia there is no negative influence on sexual potency. Nevertheless, should occasional difficulties with sexuality arise, spinal anesthesia does not have to be considered a primary causative factor.

Effects of isoflurane on the DNase I activity in an isolated enzyme preparation and on the DNase I-G actin complex.

Effects of isoflurane on the DNase I activity in an isolated enzyme preparation and in the DNase I-globular (G) actin complex were investigated. DNase I, DNase I-G actin complex, and G actin were exposed to various (0.2-4.0 vol%) isoflurane concentrations for 180 min. Thereafter, DNase I activity was determined. DNase I activity was inhibited in relation to time and concentration of isoflurane exposure. At concentrations ranging from 0.2 to 1.0 vol% of isoflurane inactive DNase I was activated in the DNase I-G actin complex. The DNase I inhibitor G actin showed a reduced capability to inhibit DNase I following isoflurane exposure. Albumin can inhibit the DNase I inactivation possibly by competition in the reactions between DNase I/albumin and isoflurane. After exposure to isoflurane the absorption maximum of DNase I was identical with the absorption maximum of heat-denatured DNase I. The results suggest a mechanism by which isoflurane may affect DNA in an indirect way at concentrations to which the patient is exposed during clinical anesthesia.

The effects of enflurane on the DNase I activity in an isolated enzyme preparation and in the DNase I-G actin complex.

The effects of enflurane on the DNase I activity in an isolated enzyme preparation and in the DNase I-G actin complex were investigated. DNase I, DNase I-G actin complexes and G actin were exposed to various (0.2-5.0 vol.%) enflurane concentrations for 180 min. Thereafter, DNase I activity was determined. Compared with controls, DNase I activity and the inhibitory capacity of G actin for DNase I were not affected by enflurane. However, there was a shift in the absorption maximum of DNase I after exposure to enflurane. The results suggest that enflurane alters the DNase I conformation without inhibition of DNase I function. In clinical subjects, side effects of anaesthesia can occur by interactions between hydrophobic anaesthetics and the hydrophobic groups of amino acids in proteins.

[No effect of injection volume on sensory and motor blockade in isobaric spinal anesthesia]. / Kein Einfluss des Injektionsvolumens auf die sensible und motorische Blockade der isobaren Spinalanaesthesie.

The authors were interested to find whether the course of sensory and motor blockade in isobaric spinal anesthesia was determined by the dose or the volume of the anesthetic agent. In a randomized double-blind study in 60 patients, each underwent isobaric spinal anesthesia with 17.5 mg bupivacaine. In three groups of 20 patients, this dose was administered as 3.5 mg bupivacaine 0.5%, 7 ml bupivacaine 0.25% or 10 ml bupivacaine 0.175%. The development, spread and regression of sensory block (anesthesia, analgesia) and motor block (Bromage scale, rectus abdominis muscle test) were determined. The clinical data recorded in the three groups were comparable. The rate of development, the maximal spread or intensity, and the regression of sensory and motor blockade did not differ in the three groups. The only difference was that the complete regression was shorter following 10 ml bupivacaine 0.175% (P less than 0.05). It is therefore concluded that the dose, and not the volume, determines the course of sensory and motor blockade of isobaric spinal anesthesia.

[Differentiating between intensity and frame of mind in patients with chronic pain.]. / Differenzierung zwischen Schmerzintensität und Gemütsverfassung bei chronischen Schmerzpatienten.

Chronic pain is a complex experience that may change the life of the patient totally. Being influenced by numerous factors, communication between the physician and the patient on such a complex experience is not always easy. The question of how patients differentiate between the intensity of their pain and their mood state was investigated in our study with the use of a new method-the dolorimeter- in 200 patients. Our results showed that the new method is appropriate to evaluate the intensity of pain in patients with chronic pain, but no to explore the patient's psychological state. The patients' assessment showed clearly that they preferred the dolorimeter to evaluate their pain intensity while they preferred a verbal scale (Profile of Mood States) to describe their mood state.

[Measurement of pain.]. / Schmerzerfassung : Beschreibung einer neuen Methode.

The aim of this study was to evaluate a new modified visual analog scale, called the dolorimeter, together with a verbal rating scale (VRS) and a linear visual scale (VAS), in the measurement of acute postoperative pain. The scales were evaluated with reference to their sensitivity, reliability and validity, and correlation. During the study 200 patients 11-70 years of age (125 men, 75 women) were interviewed after orthopedic surgery to ascertain the intensity of the pain. We had the patients judge the intensity of pain before and 1 h after giving analgesics by using the dolorimeter, VRS, and VAS. At the end of the examination, we asked the patients whether the pain had decreased or not which method they preferred, and why they preferred this method. The results of this interrogation proved that the sensitivity of the VRS is low; its parameters overlap greatly on the analog, scale, and it is therefore too rough to be a sufficient measurement of pain. On the other hand, the high sensitivity of the two analog scales which patients can use to determine their individual pain intensity proved to be much more sensitive. All three methods correlated statistically; the highest correlation coefficients were found between the analog scales VAS and the dolorimeter. Because the dolorimeter is clearly preferred to the other methods, especially by elderly patients, we came to the conclusion that the dolorimeter is less abstract than the VAS and more practical to handle.

[O.15 mg Intrathecal buprenorphine applied for postoperative analgesia. A clinical double-blind study]. / Buprenorphin 0.15 mg intrathekal zur postoperativen Analgesie. Eine klinische Doppelblindstudie.

In a double-blind, randomized study of 29 patients who underwent orthopedic procedures we studied the additional effect of intrathecal buprenorphine on isobaricpinal anesthesia and postoperative analgesia. The injections were 20 mg tetracaine (19 patients) or 20 mg tetracaine plus 0.15 mg buprenorphine (10 patients). In both groups the drugs were contained within a total volume of 4 ml cerebrospinal fluid. Progression and regression of the sensory blockade of spinal anesthesia were estimated with pinprick; the motor blockade was judged by the Bromage scheme. Postoperative pain was evaluated by the patients using an analogue scale after Scott and Huskisson. Arterial blood gases, respiratory rate, blood pressure, and heart rate were measured and other side-effects determined. Both groups were comparable in age, body weight, height and duration of operation (Table 1). The addition of buprenorphine elevated the sensory blockade by three segments both during spread and regression of anesthesia (Figs. 1, 2). Postoperative analgesia was better up to 8 h after injection (p less than 0.05), after 8 h pain levels were equal in test and control groups (Fig. 3). After buprenorphine patients became aware of pain sensation 13 h after injection; in the control group the pain-free interval lasted only 9 h (p greater than 0.05). There were no differences in the need for postoperative analgesics between both groups. The respiratory rate was lower during the whole period of observation (p less than 0.05). The mean values for PaCO2, pH and BE were similar in both groups (Fig. 4). PaO2 was elevated in the buprenorphine group. There was no essential alteration of blood pressure after buprenorphine. The pulse rate, however, was slightly diminished.(ABSTRACT TRUNCATED AT 250 WORDS)

[Midazolam (Dormicum) as oral premedication for local anesthesia]. / Midazolam (Dormicum) zur oralen Prämedikation vor Regional-Anaesthesie.

Good physician-patient rapport and an anxiolytic, sedative, and amnesic premedication are necessary for comfortable, stress-free surgery under local anesthesia. Sufficient experience exists with the intramuscular and intravenous administration of the new benzodiazepine midazolam (Dormicum), while knowledge relating to its oral administration is still scant. Therefore, in a randomized double-blind study midazolam was investigated for oral premedication prior to local anesthesia: two dosages of midazolam were studied and compared with diazepam and placebo. One hour prior to ophthalmic surgery under local anesthesia, four randomized groups of 30 patients each, received a tablet of 7.5 or 15 mg midazolam, 10 mg diazepam, or a placebo. Following this medication, the anxiolytic, sedative, amnesic, and side-effects were determined at defined points of time during the day of surgery and the 1st postoperative day. Anxiolysis was determined using the "state-trait anxiety inventory (STAI)" of Spielberger et al.; sedation was assessed according to Pandit et al.; amnesia was determined by recall of picture cards which had been presented to the patients 50 min after premedication; and patients were asked about 13 side-effects typical of benzodiazepines in a standardized way. Anxiety increased little following the placebo; it decreased significantly following 10 mg diazepam and more markedly following 7.5 and 15 mg midazolam. Sedation increased little following the placebo; it increased more and similarly 50 min after the benzodiazepines; after 90 min the sedative effect was most marked for 15 mg midazolam. However, sedation was of shorter duration after midazolam than after diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of spinal CSF dynamics on the subarachnoid diffusion of a substance applied close to the spinal cord]. / Der Einfluss der spinalen Liquordynamik auf die subarachnoidale Ausbreitung rückenmarksnah applizierter Substanzen.

In order to better understand the effects and side effects of intraspinal administration of morphine we studied the rostral spread of a comparable substance within the cerebrospinal fluid (CSF). This study was performed in connection with nuclear medical diagnostics ruling out possible rhinorrhoea or disturbances of CSF-circulation in 14 patients: Following lumbar intrathecal injection of the tracer 111-Indium-DTPA, the radioactivity over the medulla oblongata was measured continuously for 2 1/2 hours with a single probe scintillation counter; thereafter the distribution of activity over the total spinal canal was determined; finally the spread of activity was registered with the gamma scintillation camera in the 3rd, 24th and 48th hour. The diffusion of the tracer was followed in a model of the subarachnoid space. A few minutes after injection, activity over the medulla oblongata could be detected; initially it increased markedly, later less so; at the end of the 2 1/2 h observation time, approximately 8% of the total activity had reached this level. The timing of activity increase and the peak activity over the medulla oblongata varied between the individuals. Up to 48 hours the activity continued to shift from the spinal canal to the endocranium. Diffusion played a secondary role. These results are further evidence that morphine is transported cephalad within the CSF rather quickly and may act on cervical spinal cord and brainstem.

[Comparison of prilocaine 2% versus lidocaine 2% with adrenaline in peridural anesthesia. A clinical double-blind study]. / Vergleichende Untersuchung mit Prilocain 2% bzw. Lidocain 2% jeweils mit Adrenalin zur Periduralanaesthesie. Eine klinische Doppelblindstudie.

Sensory and motor blockade as well as formation of methaemoglobin were investigated under controlled double-blind conditions following epidural anaesthesia with prilocaine 2% or lignocaine 2%, each with adrenaline 1:200,000. 20 ml (= 400 mg) of these two solutions were administered to two groups, each consisting of 10 patients. Sensory blockade was tested with the pin prick method, motor blockade with the Bromage score and the rectus abdominis-muscle (RAM)-test. Venous methaemoglobin was determined before and 2,5 h after administration of the local anaesthetic. Times of onset of sensory blockade and motor blockade, as obtained with the RAM-test, were slightly earlier following lignocaine. The intensity of sensory blockade was more marked following prilocaine. The duration of action was somewhat longer following prilocaine. Methaemoglobin always increased following prilocaine, but not following lignocaine. One patient had an increase of methaemoglobin from 0.8 rel% before to 13.8 rel% after administration of prilocaine. The differences of sensory and motor blockade are of secondary importance for clinical practice; while lignocaine shows higher toxicity to the central nervous and cardiovascular system, prilocaine forms methaemoglobin.