Conjunctival provocation test: high clinical reproducibility but little local temperature change. Ten atopic and five healthy individuals participated in eight conjunctival provocation tests (CPT) in an effort to improve the CPT procedure. Results were evaluated by thermography and the use of conventional criteria under various conditions. Due to considerable spontaneous variability and change in temperature already after instillation of diluent, thermography was not sensitive enough to identify positive CPT results. The precision of the CPT was within one 10-log step in all tests and in all patients. Itching appeared before erythema in 83% of subjects and within 5-8 min after instillation of the allergen. This was true also when only one eye was used for the CPT. We therefore conclude that the CPT procedure can be used in one eye every 5-8 min when indicated in clinical routine and that it is a safe, precise method.


Pulmonary tuberculosis in patients treated with inhaled beclomethasone. Inhaled beclomethasone dipropionate (BDP) has been used with few side-effects in the treatment of bronchial asthma for 2 decades. Until now the manifestation of tuberculosis (TB) in patients on inhaled BDP has not been reported. Eight patients with allergic asthma, of a total of 548 asthmatics (1.46%) seen over a 2-year period, developed active TB following the use of inhaled BDP. All were sputum-positive for acid-fast bacilli (AFB) on smear and/or culture, all responded well to a combination of anti-TB drugs, and none showed evidence of immunological or pituitary-adrenal suppression. Two patients agreed to a repeat administration of BDP; both developed TB again within 2 weeks and are again on anti-TB treatment.


Early and late-phase asthmatic reactions: a hypothesis. Why might a foreign particle, exercise or fog inhalation precipitate two asthmatic reactions? It is reasonable to suspect that asthmatic patients develop two reactions because healthy subjects may also develop two physiological responses to the same stimuli. This suggestion is supported by the observation that living things have developed adaptative systems to confront either sudden or persistent changes in the environment or within the internal milieu. The results of several studies suggest that the early phase response (EPR) usually involves cells which are normal residents of the respiratory epithelium (mast cells) and pre-formed substances (histamine), whereas cells participating in the late phase reaction (LPR) are recruited from the circulation (eosinophils, basophils and T cells). Up to now most of the studies of the EPR and LPR have been addressed to detecting a cell or metabolite abnormality. This simplistic approach would probably not improve the knowledge of the mechanisms involved in asthmatic responses. Since the presence of isolated or dual responses seems to depend on the intensity and duration of the stimuli it is reasonable to suspect that EPR and LPR are the result of an excessive adaptative response of the bodies of asthmatics to sudden and prolonged/strong stimuli, respectively.


Nasal memory T lymphocytes capable of producing IL-4 in the allergic reaction. This paper reports the presence of memory T cells in the nasal mucosa of allergic patients. The demonstration of CD4+/CD29+ (CD4+/CD45RO+) T lymphocytes, which are capable of interleukin-4 production, can indicate a complementary cell-mediated regulatory mechanism for mast cell proliferation and IgE synthesis in human nasal allergy. No substantial IgE production can be obtained in the absence of IL-4. Therefore, the existence of IL-4 producing cells on site in the nasal mucosa of allergic subjects probably implies a complementary interaction between cytokines and different immunocompetent nasal cells in the regulation of B cells and IgE synthesis.


Hypersensitivity to trimethoprim.

Hypersensitivity to trimethoprim. We present two patients who experienced life-threatening immediate reactions and one patient who developed generalized urticaria following oral administration of trimethoprim (TMP) and sulfamethoxazole (SMX) combination. Skin prick tests with TMP were positive in the three patients. No patients reacted to skin prick tests with SMX. No significant levels of IgE antibodies to TMP were found by RAST in the serum of the patients. Normal subjects used as controls did not react to any of these tests. Single-blind, placebo-controlled oral challenges were positive with TMP and negative with SMX in all patients. These results suggest that the three patients developed type I hypersensitivity reactions to TMP. In our patients skin prick tests with TMP were useful in TMP hypersensitivity diagnosis.


Recurrent pericarditis: a rare complication of allergen immunotherapy. We report on a 29-year-old woman suffering from hay fever due to grass and olive tree pollens. She developed recurrent pericarditis during her first course of immunotherapy with an alum-adsorbed pollen extract. A causal relationship was established between the allergen injections and the acute pericarditis episodes on two consecutive occasions, which presented with blood eosinophilia. Blood cultures and serological tests for microorganisms were negative. There were no signs of autoimmune disease or systemic vasculitis. To the best of our knowledge, allergen immunotherapy-induced pericarditis has not been previously reported.


[Skin melanomas of the head and neck: therapeutic value of cervical lymphatic dissection]. There is a conflicting evidence regarding the value of a regional lymph node dissection in early clinical stages of head and neck Melanomas. The AA. reviewed the clinical histories of 500 patients presenting a malignant melanoma localized in the head and neck region and treated in their Hospital between 1967 and 1987. Results showed that the prophylactic neck dissection is necessary for lesions between 0.75 and 1.5 mm in thickness, whereas in tumours with a depth of invasion greater than 1.5 mm the outcome of the disease is not improved by the prophylactic neck dissection.


[Review of ORL manifestations in AIDS. Case series at the Arnau de Vilanova Hospital, Lleida]. The majority of patients suffering AIDS-syndrome present with ENT aspects, being the most prevalent lateral adenopathy of the neck firstly, followed by thrush, pharyngo-esophageal candidiasis and herpes simplex. Differences regarding the distribution in percentage in risk groups are signaled by the AA. between their own statistics and those of the americans writers.


[Sarcoidosis as a cause of paralysis of the recurrent laryngeal nerve. Presentation of a case]. A patient with Sarcoidosis presented with hoarseness in the course of the disease, caused by mediastinal adenopathies compressing the left recurrent laryngeal nerve. Vocal cord paralysis is an unusual complication of sarcoidosis. We have found in the consulted worldwide bibliography on the subject only four cases of sarcoidosis with mediastinal enlarged lymph nodes causing compression of the left recurrent nerve and subsequently vocal paralysis.


[Diagnostic and therapeutic management in malignant neck tumors of unknown origin. Our experience in the last 5 years]. The AA. analyzed 37 cases of metastatic neck cancer from unknown primary sites treated at the Oncologic Unit from 1986 to October 1991. This group account for 7 percent of total cervicofacial tumors seen. Factors that affected survival include the symptoms of consult, the control of cervical disease after treatment and surgery. The survival was 50 percent at two years and the rate of neck disease control amounted to 57.7 percent.


[Iatrogenic foreign body of the esophagus]. The AA. report the case of a 79 years-old patient suffering an esophageal stenosis managed with intubation (Celestin's tube). This instance shows the good tolerance of the prosthesis by the patient as well as the undue of its stay. And by the way, the simplicity of its removal with a rigid esophagoscope, as was the present case. The paper contemplates a brief review of the indications for esophageal intubation and possible complications when dealing with organic stenosis of the esophagus.


[Characteristics of subglottic and glottic-subglottic carcinomas]. The 14 subglottic and glottic-subglottic carcinomas treated in the Otolaryngologic Department of the AA. between 1984 and 1989 are reviewed. Check of literature about subglottic carcinomas.


[Prognostic value of histopathological parameters in epidermoid carcinoma of the vocal cords in T-1 stage]. In this paper are studied macro- and microscopically, 14 histopathologic parameters belonging to 37 patients suffering from vocal cord squamous cell carcinoma, stage T-la. All patients were submitted to cordectomy and followed up during a 5 years term, as a minimum. A discriminant analysis was performed in order to establish the relation between histopathologic parameters and local failure. Only two of them were statistically significative (p < 0.01): the structural type and the differential degree of tumor cells. The histopathological study allowed the prediction of 82 percent of local recurrences.


[Prolactinoma as atypical tumor of the nasopharynx]. The paper deals with a Prolactinoma case appearing as a swelling of the rhinopharynx, which developed cephalgia related to the nasal blockade as solitary symptom. Recite of the main clinical characteristics, diagnostic process and therapeutics of this condition. The differential diagnosis with other tumors situated in the sellar space or in the rhinopharynx, is also considered.


[Thyroid gland stimulation test in Reinke's edema. A study of 28 patients]. In order to clarify the correlation between Reinke's oedema and thyreopathies, we performed a study on 28 patients affected by polypoid degeneration of the vocal cords. Every patient had a complete check-up concerning the otorhino-laryngological either endocrinological aspects, in particular blood laboratory tests including the routine ones and RIA for T3, T4, FT3, FT4, hGT, antibodies and TSH under stimulation of the TRH. 22 patients showed to be affected by subclinical hypothyroidism; 5 patients did not show a substantial increase of the TSH to the TRH stimulation; one patient was affected by hypothyroidism. Our experience clearly shows that subclinical hypothyroidism is the organic pathology more frequently correlated with Reinke's oedema.


[Histiocytoid hemangioma].

[Histiocytoid hemangioma]. Angiolymphoid hyperplasia with eosinophilia is a multisystemic disease characterized mainly by cutaneous nodules, vascular proliferation with atypical histiocytoid endothelial cells and numerous eosinophiles, often with blood eosinophilia. It use to recur "in situ" and persist after surgical excision. The AA. review the literature about this rare tumor and present the clinical and histopathological features of a case of Angiolymphoid hyperplasia with eosinophilia of the tongue.


Does the laryngeal mask airway compromise cricoid pressure? The laryngeal mask airway was inserted in 10 cadavers. At postmortem the chest was opened and an infusion set primed with a dilute barium solution was inserted into the oesophagus and ligated in place. A cricoid force of 43 N was then applied and the infusion set was positioned so that when the clamp was opened it generated a pressure of 7.8 kPa within the oesophagus. The cricoid pressure was able to stop the flow of fluid into the oesophagus. This demonstrates that cricoid pressure is effective in preventing reflux at intragastric pressures which are encountered clinically and the presence of the laryngeal mask airway does not compromise this.


Unconscious learning during anaesthesia. Forty-three surgical patients were, during general anaesthesia, presented (via headphones) with either statements about common facts of some years ago (group A), or new verbal associations, i.e. the names of fictitious, nonfamous people (group B). None had any recall of intra-operative events. In a postoperative test of indirect memory, patients in group A answered more questions about the 'common facts' correctly than those in group B (p < 0.005), which reflects the activation of pre-existing knowledge. Furthermore, patients in group B designated more 'nonfamous names' as famous (thus falsely attributing fame) than patients in group A (p < 0.001), which demonstrates that information-processing during anaesthesia can also take place as unconscious learning.


The influence of arm position and cardiac output on bolus clearance from the arm. The importance of arm position and cardiac output on the clearance of a bolus injection from the arm was assessed in 63 patients using technetium-99m. Injections were made in the left arm which was either adducted, abducted or adducted with the forearm flexed over the lower chest. The clearance of isotope was assessed by measuring the amount of radioactivity remaining in the arm at 10 s intervals and calculating it as a fraction of the injected dose. The clearance of Tc-99m was significantly faster and more complete from the abducted arm than from the adducted arm. There was no correlation between clearance and cardiac output.


Cannulation of the internal jugular vein. The very high approach. A new approach to internal jugular vein catheterisation in the neck (the 'very high' approach) was used in 335 patients over a 12-month period. The success rate was 100% and there were no complications. It proved to be an easy technique to learn and may be particularly useful in difficult and emergency situations.


Six months of shivering in a district general hospital. Of 2595 patients admitted to a recovery room in Derbyshire Royal Infirmary over a 6-month period, 164 (6.3%) shivered postoperatively. Data regarding the anaesthetic techniques to which these patients had been subjected were gathered from the Derby Anaesthetic Audit System. Subsequent analysis demonstrated the importance of a number of factors that led to shivering, including male gender, anaesthetic techniques involving spontaneous ventilation, and anticholinergic premedication. The administration of pethidine, alfentanil or morphine intra-operatively reduced the incidence of shivering postoperatively.


The laryngeal mask airway in infants.

The laryngeal mask airway in infants. Clinical and fibreoptic assessment of positioning of the size 1 laryngeal mask airway was performed in 50 infants. A clinically patent airway was obtained in 47 patients at the first attempt, but perfect positioning, as assessed by fibreoptic laryngoscopy, was found in only 22 instances. Despite an airway initially patent, delayed airway obstruction occurred in 12 infants. It is concluded that clinical airway patency does not guarantee ideal positioning of LMA in infants, and that care should be taken to ensure continued airway patency because of the tendency of the LMA position to deteriorate in this group of children.


Profound hypotension following intravenous codeine phosphate. Three case reports and some recommendations. Three adults are described who developed life-threatening hypotension following intravenous codeine phosphate. It is recommended that codeine phosphate should not be given intravenously to adults.


Repeated unilateral epidural blockade.

Repeated unilateral epidural blockade. Unilateral epidural analgesia occurring in a parturient three times in successive pregnancies is reported. Possible causes are reviewed, and clinical and radiological evidence in support of the most likely explanation are presented.


Profound desaturation following vomiting on induction. A case for routine pre-oxygenation. Whilst participating in a clinical trial, a patient vomited during induction of anaesthesia. Subsequent analysis of the continuous record of oxygen saturation showed precipitous desaturation.


Anaphylactoid reaction to propofol.

Anaphylactoid reaction to propofol. A case of an anaphylactoid reaction following the induction of anaesthesia is reported. Subsequent intradermal testing suggested propofol to be the causative agent.


Anaesthetic machine checking practices. A survey. Forty anaesthetists, of all grades, were interviewed without prior warning and questioned about the checks they had performed on their anaesthetic equipment before use. The results reveal that a substantial percentage (up to 41%) of anaesthetists perform inadequate checks. Furthermore, of those that do, few follow the Association of Anaesthetists of Great Britain and Ireland's recent guidelines.


Evaluation of the Level 1 Hotline blood warmer. The Level 1 Hotline warmer is a new type of fluid warmer, developed specifically for use with low flows. It was found to be effective at flows between 1 ml.min-1 and 75 ml.min-1.


The extensometer--use in spontaneously ventilating awake volunteers. The extensometer is a new device with potential in the field of respiratory pattern analysis. This paper describes the physical principles upon which the extensometer depends and also assesses its performance as a noninvasive respiratory monitor in respect of its ability to measure tidal volume and to determine obstructive breathing patterns in awake volunteers in the supine position over a limited time period. Further developments of the device are outlined and the current status of torso transducers in anaesthesia and intensive care are discussed.


An evaluation of the gum elastic bougie. Intubation times and incidence of sore throat. This study was designed to evaluate the routine use of a gum elastic bougie for tracheal intubation. The median time to intubation with the gum elastic bougie while simulating an 'epiglottis only' view was only 10 s longer than the time taken during conventional intubation with an optimum view. Three of the patients required a gum elastic bougie-assisted intubation after attempts at conventional visual intubation had failed. There was no significant difference in the incidence of postoperative sore throat and hoarseness between the two groups. We recommend that anaesthetists should use the gum elastic bougie whenever a good view of the glottis is not immediately obtained.


The Mancunian way.

The Mancunian way. The authors have examined early records of the administration of ether anaesthesia in Manchester and the lives of several medical men involved in these events. Charles Strange, a dentist and chemist, in a letter to the Manchester Guardian published on 14 January 1847, described a self-administration of ether for dental extraction, but George Bowring, a surgeon, subsequently claimed the first anaesthetic administered by a doctor in Manchester. The merits of these claims are discussed in the light of the circumstances surrounding these events.


Plasma concentrations of flumazenil during liver transplantation. Six patients were given flumazenil (1 mg) during the anhepatic period of liver transplantation. We found higher mean plasma flumazenil concentrations during the anhepatic period than occurred in patients with hepatic cirrhosis. It was only possible to measure plasma flumazenil concentrations in four patients after revascularisation of the donor liver. Of these, elimination half-lives could be calculated in only three patients (normal in one and prolonged in two). In these two patients the elimination half-lives were prolonged to the same extent as in patients with fulminant hepatic failure. The changes during the anhepatic period occur because the liver contributes to the central volume of distribution. After revascularisation of the donor organ the prolonged half-lives indicate that transplanted livers may not recover normal metabolic functions immediately.


Local anaesthetic: does it really reduce the pain of insertion of all sizes of venous cannula? A recent study performed in this department showed that a subcutaneous injection of local anaesthetic was significantly less painful than the insertion of a 22-gauge venous cannula. However, our colleagues remained sceptical that local anaesthetic infiltration would eliminate the pain of cannulation. Consequently a further study was undertaken to compare the pain of cannulation with and without the use of local anaesthetic. The results show that pain of cannulation is significantly (p < 0.003) reduced after subcutaneous infiltration with 1% lignocaine when compared to cannulation without local infiltration. Persistent discomfort at the site of cannulation was eliminated by the use of local anaesthetic.


Day case laparoscopy. A comparison of two anaesthetic techniques using the laryngeal mask during spontaneous breathing. Forty women undergoing day case laparoscopy were randomly allocated to receive either an inhalational or total intravenous anaesthetic. All patients breathed spontaneously through a Brain laryngeal mask. There were no clinically significant cardiovascular or respiratory differences between the two techniques. No episodes of clinical aspiration occurred and there were no peri-operative arrhythmias. We conclude that spontaneous respiration with a laryngeal mask is a safe and effective method of anaesthesia for laparoscopy provided certain guidelines are followed.


An audit of induction of anaesthesia in neonates and small infants using pulse oximetry. The frequency and severity of hypoxaemia during induction of anaesthesia in neonates and small infants at the Norfolk and Norwich Hospital, a district general hospital, was compared, using pulse oximetry, with that of the nearest specialist hospital, the Queen Elizabeth Hospital for Sick Children in London. There were differences in staffing and the choice of anaesthetic techniques between the hospitals. One third of the patients in both hospitals experienced desaturation of more than 5% (moderate or severe hypoxaemia) at one or more recordings during induction. The highest incidence of hypoxaemia was associated with awake intubation. There was no statistical difference in the incidence or severity of hypoxaemia between the hospitals. In the district general hospital, moderate or severe hypoxaemia of greater than 30 s duration was more likely if an anaesthetist with a regular paediatric operating list was not present at induction (p < 0.01).


Patient controlled analgesia--assessment of machine feedback to patients. The effect of varying patient-controlled analgesia machine feedback was studied in two groups of patients following a standard surgical procedure using patient controlled analgesia with morphine to control postoperative pain. Analgesic demands, morphine consumption, pain scores and side effects were compared and comments from the patients were noted. There was no significant difference between those whose machines signalled only successful demands compared with those who had every demand acknowledged. The patients in the former group appreciated the extra input and the nursing staff found this machine configuration to be less disturbing to the ward. There was no evidence of a clinically useful placebo effect in the latter group.


Spinal anaesthesia for urological surgery. A survey of failure rate, postdural puncture headache and patient satisfaction. A survey was conducted on 100 consecutive patients who underwent spinal anaesthesia in our urology operating theatres. Details of the spinal technique were recorded in the operating theatre. In 25% of patients, more than one attempt at subarachnoid puncture was required and 16% of this group went on to require general anaesthesia. The patients were visited between 24 and 48 h postoperatively by one of the authors. On questioning, 24% of patients reported a headache, which had the characteristics associated with dural puncture; 62% of these headaches were described as moderate or severe and lasted between 12 and 24 h. Patients were significantly (p < 0.05) more likely to develop a postdural puncture headache if more than one attempt at subarachnoid puncture was made.


[The action of S-(+)-ketamine on serum catecholamine and cortisol. A comparison with ketamine racemate]. The S(+)-isomer of ketamine has about twice the anaesthetic potency of the commercially available racemic mixture of ketamine. It is assumed that the known side-effects of ketamine are significantly reduced when administering half the usual dose with the same pharmacodynamic effect [17, 25]. The aim of the present study was to determine the haemodynamic effects, the catecholamine and cortisol plasma levels after administration of equally potent doses of S-(+)-Ketamine and racemic mixture of ketamine. In addition, the effect of premedication with i.v. midazolam was assessed. METHOD. After approval by the ethics committee and written informed consent, 30 healthy male volunteers were randomly allocated to three groups (n = 10). Group 1 received 2 mg/kg ketamine racemate, group 2 1 mg/kg S-(+)-Ketamine, and group 3 1 mg/kg S-(+)-Ketamine 5 min after i.v.-premedication with 0.1 mg/kg midazolam. Non-invasive blood pressure (BP) and heart rate (HR) were continuously recorded. Blood samples were drawn 7 min before, and 2, 4, 8, 16, 32, 64 and 128 min after drug administration. Plasma epinephrine and norepinephrine (NE) levels were determined by HPLC and cortisol plasma levels by RIA. Data were analysed with the Kruskal-Wallis test (P < or = 0.05) for differences between groups. RESULTS. HR and BP showed a significant rise after injection of racemate and isomer, without any significant differences between groups. This was also seen for norepinephrine and cortical plasma levels. Epinephrine levels, however, differed between groups, showing a significant rise after racemate compared to isomer. Premedication with midazolam, in contrast, blunted major haemodynamic and hormonal changes. DISCUSSION. The haemodynamic changes did not differ between the racemate and isomer group despite a reduced isomer dose. HR and BP rise were similar, although epinephrine levels were significantly lower after isomer than racemate. Hence we assume that the increase in the haemodynamic parameters were mainly caused by NE. Midazolam apparently prevented the centrally mediated sympathetic stimulation caused by ketamine and its isomers. Therefore, i.v. premedication with midazolam should be applied when racemate or isomer is used, especially in high-risk cardiac patients.


[Studies using S-(+)-ketamine on probands. Computerized EEG-analysis and transcranial Doppler ultrasonography]. It has been shown in the recent literature that the anaesthetic potency of the ketamine isomer S(+)-ketamine is twice that of the racemic mixture used in clinical practice. METHODS. With approval of the local ethics committee, we investigated the effects of a bolus injection of 2 mg/kg racemic ketamine or 1 mg/kg S(+)-ketamine, respectively, on the electroencephalogram (EEG) and transcranial Doppler sonography (TCD) of the middle cerebral artery in ten healthy volunteers by means of a randomised, double-blind, cross-over design. Spectral edge frequency, total activity and activity of the alpha, beta, delta, and theta bands were assessed by bihemispheric leads using the Neurotrac system before (MZP 1) and 1, 3, 5, 10, 15, 30, 60, and 120 min after injection (MZP 2-9). Simultaneously, systolic (Vs-MCA), diastolic (Vd-MCA), and mean (Vm-MCA) blood flow velocities in the middle cerebral artery and pulsatility (Vs-Vd)/Vm were recorded. Statistics consisted of two-dimensional analysis of variance with P < 0.05 considered as significant. RESULTS. Racemic ketamine and S(+)-ketamine produced comparable effects on the EEG. Theta- and beta-activities increased between the 1st and 10th min after injection and returned to near baseline values thereafter. 120 min after injection, the absolute delta- and theta-activities were higher following racemic ketamine when compared to the S(+)-ketamine group. However, significant differences between the groups could not be confirmed statistically. TCD variables changed considerably in the course of the study without intergroup differences. Compared to the baseline values, Vs-MCA, Vd-MCA, and Vm-MCA increased significantly for the first 15 min after injection with a maximum at MZP 3. The pulsatility of the obtained Doppler waveform (Vs-Vd)/Vm showed significant decreases at MZP 2-4. CONCLUSIONS. In our study, both racemic ketamine and its isomer S(+)-ketamine produced similar EEG changes consistent with the data from earlier studies. However, the relative lower proportion of slow EEG activity at the end of the study might indicate a better recovery of cortical function following S(+)-ketamine than after racemic ketamine. Assuming a close relationship between cerebral blood flow velocity and cerebral blood flow, our TCD results suggest that both racemic ketamine and S(+)-ketamine will considerably increase cerebral blood flow in spontaneously breathing volunteers. Such an effect has been observed by others and, at least partly, can be explained by a concomitant increase in arterial carbon dioxide partial pressure.


[Ketamine racemate or S-(+)-ketamine and midazolam. The effect on vigilance, efficacy and subjective findings]. Ketamine is a racemic mixture containing equal amounts of optical isomers that have almost identical pharmacokinetic properties but different pharmacodynamic effects. The S-(+)-isomer of ketamine has about twice the anaesthetic and analgesic potency of the racemic ketamine preparation and is judged to induce less psychic emergence reactions and to be followed by a more rapid recovery of vigilance. The present study was designed to assess whether the S-(+)-isomer of ketamine is superior to the racemic mixture in cardiovascular characteristics, emergence reactions and cognitive functions, and whether side effects may be reduced or prevented by administration of midazolam prior to injection of S-(+)-ketamine. METHODS. Following ethics committee approval and informed consent, 30 volunteers were randomly allocated in this double-blind study to three groups of 10 each. Group 1 received 2 mg/kg bw racemic ketamine, group 2, 1 mg/kg bw S-(+)-ketamine and group 3, 1 mg/kg bw S-(+)-ketamine after premedication with 0.1 mg/kg midazolam i.v. Cardiovascular changes, state of vigilance, cognitive performance, subjective mood and acceptance of anaesthesia were assessed by means of haemodynamic routine monitoring, electroencephalography (EEG), psychometric tests and interview. RESULTS. The increases in mean arterial pressure and heart rate following the injection of racemic ketamine and S-(+)-ketamine were identical and the differences from baseline values significant after both. Premedication with midazolam ensured stable haemodynamics after injection of S-(+)-ketamine. EEG analysis displayed the characteristic changes well known from ketamine anaesthesia for both racemic and S-(+)-ketamine. The vigilosomnoscript showed an identical profile of vigilance up to 30 min after injection of both drugs. The vigilance status after 125 min was less impaired by S-(+)-ketamine than by racemic ketamine. Psychological assessment showed a prompter recovery of visual attentiveness and sensorimotor performance in the S-(+)-ketamine group. Subjective mood was judged by the volunteers to be significantly better after S-(+)-ketamine, and volunteers found S-(+)-ketamine to be more acceptable than racemic ketamine. The frequency of dreams was the same after both drugs. No unpleasant dreams were reported after S-(+)-ketamine, but one of the volunteers who received racemic ketamine had uncomfortable dreams. Midazolam prevented any unpleasant emergence sequelae. On the other hand, the cognitive performance could not be restored to the baseline values until at least 240 min after injection of S-(+)-ketamine, because of the sedative effects of midazolam. DISCUSSION. These results suggest that S-(+)-ketamine offers the advantages of faster recovery of cognitive performance, greater acceptance by the volunteers and identical depth of anaesthesia after injection of half the dose compared with racemic ketamine. The clinical use of S-(+)-ketamine therefore seems to be justified. Premedication with benzodiazepines, e.g. midazolam, is essential. The dose to be administered, however, should be carefully selected in order not to abolish the positive effect of S-(+)-ketamine on vigilance by the sedative effects of the benzodiazepine.


[The simultaneous determination of ketamine and midazolam using high pressure liquid chromatography and UV detection (HPLC/UV)]. In order to prevent some negative side effects of ketamine during "dissociative anaesthesia", this substance is regularly combined with benzodiazepines, e.g., midazolam. This study was undertaken to develop a simple and effective method for the simultaneous detection of ketamine and midazolam in plasma by high-pressure liquid chromatography with UV-detection (HPLC/UV). METHODS. The chromatographic system consisted of a 300 x 3.9-mm C18 column for reversed-phase chromatography and a mobile phase of 30% acetonitrile and 70% 0.05 M sodium phosphate buffer, pH 4.45. The flow rate was 1 ml/min. UV-detection took place at a wavelength of 210 nm. Blood samples were preferably taken from a central venous or arterial line. After plasma separation, 1 microgram (100 microliters) etidocaine was added to 1 ml plasma as an internal standard. The samples were alkalinised and extracted with ether, followed by back-extraction of the organic phase in 250 microliters 0.05 N sulphuric acid; 50 microliters of this solution was injected into the system. RESULTS. Sample preparation by trained personnel was reliable and led to adequate results. Separation of ketamine and midazolam was very satisfactory. Standard curves for both drugs were linear from 15-4000 ng/ml (ketamine r = 0.9998; midazolam r = 0.9996). Recovery rates for ketamine in plasma were above 95%, for midazolam 70%-75%. Coefficients of variation for ketamine in plasma were between 0.6% and 1.3%, for midazolam between 2.0% and 2.6%. The detection limit was lower than 5 ng/ml. CONCLUSION. The method is suitable for clinical practice and allows the simultaneous detection of two anaesthetics in wide-spread combined use.


[Cerebral effects of ketanserin. The influence on hemodynamics and brain metabolism]. Ketanserin, a 5HT2- and alpha 1-receptor antagonist, decreases blood pressure by decreasing systemic vascular resistance without causing reflex cardiac stimulation, while cardiac output remains unchanged. To date, little is known about the effects of ketanserin on cerebral haemodynamics and cerebral metabolism. According to a recently published study, ketanserin seems not to impair cerebral blood flow autoregulation in man. The present study was designed to investigate the influence of ketanserin on cerebral circulation and metabolism, and the cerebrovascular response to CO2 in man. METHODS. Twenty male patients between 44 and 67 years of age who were scheduled for coronary artery bypass surgery were randomly allocated to one of two groups. In group 1 measurements were performed after induction of anaesthesia during normocapnia (p(a) CO2 approximately 40 mm Hg) and hypocapnia (p(a) CO2 approximately 30 mm Hg). Then, ketanserin was given at a bolus dose of 0.3 mg.kg-1 followed by an infusion of 0.06 mg.kg-1.h-1 and measurements were repeated under hypocapnic and normocapnic conditions. Patients of group 2 were hyperventilated at first, then normoventilated. Afterwards, ketanserin was administered at the above-mentioned dose and measurements were again performed during normocapnia and hypocapnia. Cerebral blood flow (CBF) was measured using the argon wash-in technique. Cerebral venous blood was obtained from a catheter in the superior bulb of the right internal jugular vein. Cerebral perfusion pressure (CPP) was calculated by subtracting jugular bulb pressure from mean arterial pressure and cerebral vascular resistance (CVR) by dividing CPP by CBF. Cerebral metabolic rates of oxygen, glucose, and lactate were calculated by multiplying the arterial-cerebral venous oxygen and substrate differences by CBF. RESULTS AND DISCUSSION. Ketanserin decreased CPP by 16% to about 60 mm Hg. Cerebral blood flow remained unchanged as a result of an insignificant decline in CVR. Hyperventilation increased CVR by 32%, while CBF decreased by 27% to the same value that had been obtained during hypocapnia without ketanserin. The percentage changes in CBF per mm Hg change in CO2 were 1.45%/mm Hg (group 1 and 2.91%/mm Hg (group 2), respectively, without ketanserin and 1.98%/mm Hg and 2.22%/mm Hg with ketanserin. As CO2-responsiveness with ketanserin was higher in group 1 but lower in group 2 than without ketanserin, the direction in which ventilation was changed rather than ketanserin was responsible for these changes in CO2-responsiveness. Neither during normocapnia nor during hypocapnia did ketanserin have any effects on cerebral metabolic activity. Thus, it can be concluded that ketanserin does not impair CBF regulation and metabolism and that cerebral vascular responsiveness to hypocapnia is preserved.


[Continuous peridural anesthesia in abdominal surgery. An alternative for elderly patients]. Being advanced in years is not in itself a high risk in anaesthesia; however, altered pharmacokinetics and pharmacodynamics, mental dysfunction and the administration of anaesthetics complicate the postoperative period. In order to examine the problem of sedation in elderly patients, we studied the effects and side effects of continuous peridural anaesthesia for abdominal surgery. METHODS. On the day before surgery we inserted a peridural catheter (Perifix 400, Braun, Melsungen, FRG) between T-12 and L-4 in 52 patients in a sitting position (mean age 69.3 +/- 10.9 years) using the loss-of-resistance technique. If no signs of spinal anaesthesia became apparent, the exact position of the catheter was determined using 9 or 10 ml bupivacaine 0.5%. Next day, after premedication with atropine, pethidine or midazolam, 20-25 ml bupivacaine 0.5% was instilled through the peridural catheter. During surgery patients were sedated using a small dose of propofol. We also insufflated oxygen (2 l/min). Blood pressure, heart rate, and blood gases were monitored and electrocardiography and pulse oximetry performed. As postoperative pain therapy, we administered morphine through the peridural catheter at intervals of 8 h. For statistical evaluation we used Wilcoxon's test. RESULTS. An adequate degree of analgesia was found between T-4 and T-7 and abdominal muscle relaxation was satisfactory. Heart rate decreased by 10.3% after the administration of local anaesthetics. After surgery had begun, blood pressure decreased over a period of 30 min (systolic by 20.5% and diastolic by 14.2%) but it remained constant at this level during the rest of the operation (see Fig. 1). Neither of these side effects was significant. Oxygen saturation and blood gases were normal. During the operation, a mean dose of 325 mg propofol/h was necessary to maintain sedation. After surgery all patients were awake, suffered no pain and had complete amnesia with regard to the operation. The postoperative peridural dosage of 5 mg morphine (three times in 24 h) was very effective. Because some patients vomited we used between 50 and 100 mg tramadol (four times in 24 h) instead of morphine. Early mobilization of patients was possible and there were no pulmonary complications such as pneumonia. CONCLUSIONS. If carried out by an experienced physician, continuous peridural anaesthesia can be an alternative method in abdominal surgery for elderly patients. We see advantages in the minimal disturbance of pulmonary and mental function, in the minimal amount of sedation required and in the successful postoperative pain therapy.


[Combined sciatic 3-in-1 block. Application in lower limb orthopedic surgery]. The effects of increasing the maximum prescribed volume of 50 ml lidocaine 1% to 65 ml in a combined sciatic 3-in-1 block were investigated in 25 adult patients. The goal of the study was (1) to show possible increase in the success rate and (2) to determine if toxic plasma levels of local anaesthetic would be reached. Further more, we wanted to find out if there were any side-effects. The patients were divided into three groups according to body weight: group I, 50-69 kg (n = 7); group II, 70-80 kg (n = 9); group III, over 80 kg (n = 9). Besides clinical observation, the plasma levels of local-anaesthetic were determined over a 90-min period. RESULTS. Following the neural blockade, the obturator nerve was the first to be rendered insensitive after 4.8 +/- 3.6 min, followed by the saphenous (7.2 +/- 4.4 min) and femoral nerves, and finally by the lateral femoral cutaneous nerve after 12.5 +/- 5.3 min. Around the knees, the motor blockade was complete after 11.9 + 4.3 min and around the hips 11.2 +/- 4.6 min. Intraoperatively, one of these 25 patients needed an additional general anaesthetic, as he did not tolerate the Esmarch cuff. The postoperative analgesia in 24 patients lasted 268 +/- 55 min; the patient who had a general anaesthetic in addition had a postoperative analgesia of 315 min. Group I showed levels of mean 2.04 +/- 0.64 microns/ml, group II 1.84 +/- 0.38 microns/ml and group III 1.69 +/- 0.63 microns/ml. There were no statistical differences between the three groups. CONCLUSION. Increasing the dosage of lidocaine from 500 to 650 mg makes the block very successful without any toxic side-effects.


[Obstructive respiration disorders. An aneurysm of the ventilator tubing during general anesthesia]. Technical problems during anaesthesia are important causes of anaesthesia-related deaths and brain damage. During general endotracheal anaesthesia for ophthalmic surgery (41-year-old man, ASA 1) we observed an increase in inspiratory pressure without other clinical changes. Disconnection and ventilation with a resuscitation bag showed normal inspiratory pressures. Inspection demonstrated an obstruction due to an aneurysm of the inner layer of the inspiratory tubing. The classification of this rare blockage of ventilation differs in the literature (pressure, hypoventilation, hypercarbia). In addition, it demonstrates the principal problem of clinical decision-making during anaesthesia based on monitoring information. Strategies for responding to alarms indicating hazards of ventilation must be based on immediate restoration of sufficient ventilation, and not primarily on detecting the cause.


[Positive end-expiratory pressure (PEEP)]. PEEP has become a widely used ventilatory technique. The beneficial effects of PEEP were first described in asphyctic neonates, and it was later used in the treatment of cardiogenic pulmonary edema. Since the 1970s PEEP has been well established for the treatment of ARDS; the technique is also used for scoring the degree of severity of ARDS. Two mechanisms have been identified to explain pulmonary function and gas exchange following PEEP therapy: increasing FRC and alveolar recruitment. Both factors result in improvement in the ventilation/perfusion ratio with a consequent decrease in the intrapulmonary right-to-left shunt fraction. PEEP should be used in cardiogenic pulmonary edema as well as in ARDS; there are few contraindications. To choose the individual level of PEEP, PEEP should be titrated in 3- to 5-cm increments and its effects on haemodynamic function, pulmonary gas exchange and respiratory mechanics taken into account. In this article the effects of PEEP, its use and abuse are reviewed from a practical point of view.


Investigation of noncalcium interactions of fura-2 by classical and synchronous fluorescence spectroscopy. Several authors have reported unexpected intracellular spectra of both indo-1 and fura-2. One of the major methodological problems in the evaluation of calcium concentration using fluorescent probes is that it is assumed that only two forms of the dyes are detectable within the cells. We show in this study of fura-2 properties that this calcium probe is pH-sensitive and able to bind to cellular proteins. The excitation spectra of protonated and protein-bound forms of fura-2 exhibit a maximum in the same region as that associated with the calcium-free form (i.e., near 365 nm). The very small shift in the excitation spectra upon proton or protein binding precludes the use of classical methods to determine the spectral composition of mixtures of several forms of fura-2. We therefore used the synchronous fluorescence technique to detect the protein-bound form of fura-2 selectively, in order to assess the pH dependence of the fura-2/protein interaction. The nonspecific binding of fura-2 to proteins is reinforced at acidic pH and inhibited by calcium. The fact that the same type of interaction was found between fura-2 and poly-L-lysine suggests that it could be mediated by basic amino acids. Because of the strong overlap of the excitation spectrum of the unprotonated free fura-2 with those associated with the protonated and protein-bound forms, a cytoplasmic acidification may lead to an artifactual measurement of low calcium levels.


Factors controlling variability in measurements of the transient gene expression in electroporated protoplasts of wheat callus. Factors controlling variability in enzyme transient expression assays have been investigated in electroporated protoplasts isolated from wheat embryogenic and nonembryogenic calli. The level of variation was reduced to a minimum through the optimization of the beta-glucuronidase measurements in the pellet and the supernatant of the homogenized protoplasts, by expressing the data on the basis of the number of protoplasts found to be viable immediately before the assay and on the amount of protein in the pellet and supernatant. Protoplast separation on the basis of size was also useful in eliminating some of the variation resulting from the heterogeneity of the callus used. Efforts to partially synchronize the callus tissue by auxin starvation have not resulted in a significant decrease of this variation. Our results indicate that the level of variation in enzyme transient activity in protoplasts resulting from calli can be reduced by implementation of the experimental techniques presented here.


Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces. A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.


The measurement of negative charge content in cartilage using a colloid titration technique. A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 mu eq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.


Fluorometric assay of 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in plasma. The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery.


Simultaneous determination of sugar incorporation into glycosphingolipids and glycoproteins. We assessed inhibitors of glycosylation by simultaneous determination of [14C]Gal incorporated into glycosphingolipids and glycoproteins as well as of [3H]Leu incorporated into proteins of intact cells. After metabolic labeling in 96-well plates in the presence or absence of a test substance, cells were collected on glass-fiber filters. The lipid components were extracted from the filter and radioactivities of both extract and filter determined. The reliability of the procedure was tested with different drugs. Using the glucocerebroside synthetase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP; 5 microM), glycolipid biosynthesis was shown to be reduced to 50% in the murine T-cell EL-4 6.1 line, whereas glycosylation of proteins was not disturbed. With 0.5 microM tunicamycin, the glycosylation of proteins was 50% of that in the control. The procedure was also able to detect various specific effects: the inhibition of protein glycosylation with D-glucosamine and castanospermine, the inhibition of glycosphingolipid biosynthesis with L-cycloserine, and a slight enhancement of glycosphingolipid biosynthesis with conduritol B epoxide and castanospermine. Within a series of N-acyl homologs of PDMP the inhibitory potency increased with chain length. In contrast, these homologs were equipotent by enzymatic in vitro assays.


Quantitation of positional isomers of deuterium-labeled glucose by gas chromatography/mass spectrometry. A method for determining the site and extent of deuterium (D) labeling of glucose by GC/MS and mass fragmentography was developed. Under chemical and electron impact ionization, ion clusters m/z 328, 242, 217, 212, and 187 of glucose aldonitrile pentaacetate and m/z 331 and 169 of pentaacetate derivative were produced. From the mass spectra of 13C- and D-labeled reference compounds, glucose carbon and hydrogen (C-H) positions included in these fragments were deduced to be m/z 328 = C1-C6, 2,3,4,5,6,6-H6; m/z 331 = C1-C6, 1,2,3,4,5,6,6-H7; m/z 169 = C1-C6, 1,3,4,5,6,6-H6; m/z 187 = C3-C6, 3,4,5,6,6-H5; m/z 212 = C1-C5, 2,3,4,5-H4; m/z 217 = C4-C6, 4,5,6,6-H4; and m/z 242 = C1-C4, 2,3,4-H3. After correction for isotope discrimination and deuterium-hydrogen exchange, the D enrichment of these fragments can be quantitated using selective ion monitoring, and the D enrichment of all C-H positions can be obtained by the difference in enrichment of the corresponding ion pairs. The validity of this approach was tested by examining D enrichment of known mixtures of 1-d1-, 2-d1-, 3-d1-, and 5,6,6-d3-glucose with unlabeled glucose and D enrichment of perdeuterated glucose using these fragments. This method was used to determine deuterium incorporation in C1 through C6 of blood glucose in fasted (24 h) rats infused with deuterated water. The distribution of deuterium was similar to that found by Postle and Bloxham (1980, Biochem. J. 192, 65-73). Approximately one deuterium atom was incorporated into C5 and only 75% deuterium atom was incorporated into C2. The enrichment of C2 and C6 of glucose relative to that of water indicated that 74 +/- 9% of plasma glucose was newly formed 4 h after the onset of deuterium infusion, and gluconeogenesis accounted for about 76 +/- 7% of the glucose 6-phosphate flux.


A specific peroxidase-coupled activity stain for diamine oxidases. A specific peroxidase-coupled activity staining method for diamine oxidase (DAO) was developed. Diaminobenzidine was found to inhibit DAO and to give rise to unspecific staining. Among several other reagents 4-Cl-1-naphthol was found to be most suitable. Using specific activity staining DAO could be visualized in polyacrylamide gels as a high-molecular-weight complex, which could be dissociated by Tween 20 but not by NP-40, Triton X-100, or Chaps.


Synthesis of [2-3H-ethyl]S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and its use in covalent-binding studies. Metabolism of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) yields chlorofluorothioacetyl fluoride, which reacts with cellular proteins to form stable lysine adducts. Little is known about the subcellular localization of these protein adducts or about their role in CTFC-induced nephrotoxicity. A method for the synthesis of CTFC and other cysteine S-conjugates labeled with 3H at the S-alkyl or S-alkenyl position would be useful in studies of S-conjugate metabolism and toxicity. Reaction of L-cysteine, chlorotrifluoroethene, 1,8-diazabicyclo[5.4.0]undec-7-ene, and 3H-labeled water followed by repeated crystallization yielded radiochemically pure [3H]CTFC (235 mg, 20% yield; sp act 1.07 x 10(9) Bq/mmol), which was identical to CTFC by TLC, 1H NMR, and 19F NMR. 3H NMR revealed a doublet of triplets at 6.5 ppm with geminal and vicinal T-F couplings of 51.5 and 6.0 Hz, respectively, consistent with the proposed structure. When 2H-labeled water was used, [2H]CTFC was formed, and its structure was confirmed by 1H and 19F NMR, FAB-MS, and TLC. Analysis of renal and hepatic subcellular fractions of rats given 1, 10, or 100 mumol/kg [3H]CTFC showed a dose-dependent binding of 3H-containing metabolites to liver and kidney proteins.


An enzyme-reactor for electrochemical monitoring of choline and acetylcholine: applications in high-performance liquid chromatography, brain tissue, microdialysis and cerebrospinal fluid. A sandwich-type enzyme reactor in which the enzymes are physically immobilized in a minimal dead space between two cellulose membranes, resulting in improved sensitivity, was developed for the electro-chemical detection of choline (Ch) and acetylcholine (ACh). The reactor contains the enzymes choline oxidase with or without acetylcholine esterase, for the detection of ACh and Ch, respectively. For the HPLC analysis of Ch and ACh the detection system was coupled post column. Levels of Ch and ACh of rat striatum tissue and human cerebrospinal fluid were found to be similar to those determined with published methods. Because of low back pressure--a further advantage of the reactor--the detection system could also be directly coupled to the outlet of a microdialysis device, allowing the on-line real-time measurement of extracellular brain Ch. The versatility of the enzyme reactor for the monitoring of analytes in HPLC eluates, flow injection analysis, with or without prepurification, is emphasized. The usefulness of the reactor-detector system in biomedical applications is illustrated by the measurement of increases of rat striatal extracellular Ch following cardiac arrest.


A potential pitfall in protein kinase assay: phosphocellulose paper as an unreliable adsorbent of produced phosphopeptides. The ability of phosphocellulose paper to retain phosphorylated peptides containing basic amino acid residues was investigated. Some peptide substrates that are commonly used for three different protein kinases were tested. The adsorption onto phosphocellulose paper was strongly dependent on the amino acid composition of the peptides. None of the phosphopeptides studied was adsorbed completely, the amount bound varied from 7 to 93%. Phosphopeptides containing two basic amino acids each differed remarkably in the degree of binding to the phosphocellulose paper (40% RRASVA, 60% FRRLSI, and 80% HRASV was bound). The results presented here indicate that data from phosphorylation experiments obtained so far for different peptides using the phosphocellulose paper method should be judged with caution.


Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells. The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT.


Quantification of peptide aldehyde ligands immobilized for the affinity chromatography of endopeptidases. A method is described for quantification of aldehyde and aldehyde semicarbazone groups attached to an insoluble matrix. Semicarbazones are converted to aldehydes, and the aldehydes are coupled with 4-phenylazoaniline. The excess reagent is washed off, and the remainder then displaced with salicylaldehyde. The quantity of the phenylazoaniline/salicylaldehyde adduct is determined spectrophotometrically, allowing the calculation of the amount of aldehyde or semicarbazone per unit volume of the matrix. Analyses by the new method show that four matrices offered commercially for this type of immobilization differ greatly in coupling capacity and stability of the conjugate under conditions of affinity chromatography.


Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content. The bicinchoninic acid (BCA) assay method for the determination of protein has been investigated for its utility in measuring the protein content of plasma lipoproteins. Although other methods, principally those based on the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275) have been extensively used for this purpose, the tolerance of the BCA method to many commonly encountered detergents and buffers offers a definite advantage over the Lowry-based methods. In this study, lipoprotein protein values obtained by the BCA method were compared to a standard modification of the Lowry et al. procedure since this assay forms the basis of much of the relevant literature. The standard BCA assay was found to overestimate the protein content of very low density lipoprotein by approximately 70% and low density lipoprotein by approximately 30%; high density lipoprotein values compared favorably. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions. This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis. In the presence of this detergent, BCA assay measurements for these three lipoprotein fractions were 97, 90, and 98%, respectively, of the reference assay values.


Ion-spray tandem mass spectrometry in peptide synthesis: structural characterization of minor by-products in the synthesis of ACP(65-74). Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65-74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Bly-OCH2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF/p-cresol for 1 h at 0 degrees C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65-74). The significant minor component was apparently from incomplete deprotection of Asp70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp70, succinimide formation at Asn73, acylation of the Tyr71 side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods.


Synthesis and properties of N-bromoacetyl-L-thyroxine. A one-step bromoacetylation of L-thyroxine (T4) produces N-bromoacetyl-L-thyroxine (BrAcT4) in good yield. The reaction product is best purified by high-speed countercurrent chromatography. While HPLC is satisfactory only for purification of microgram and submicrogram quantities, amounts ranging from about 1 ng to 1 g of BrAcT4 can be processed by high-speed countercurrent chromatography (HSCCC), a method which we have previously used for the purification of N-bromoacetyl-3,3',5-triiodo-L-thyronine (BrAcT3). Operating conditions for the one-step synthesis of BrAcT4 and BrAcT3 differ due to differences in solubility and reactivity of the two hormones. BrAcT4 purified by HSCCC and shown to be pure by analytical HPLC has been characterized by alpha max and epsilon max in the near and far uv in several solvents, mass spectrum, 1H NMR spectrum, TLC in three solvent systems, retention time in reverse-phase HPLC (C18) in relation to the retention times of two internal standards, 3,3',5-triiodo-L-thyronine and T4, and melting point. Corresponding data for BrAcT3, not previously reported, have also been determined. The described procedure can provide not only substantial amounts of highly purified BrAcT4 for competition studies, but also 125I-labeled BrAcT4 of high specific activity for affinity labeling. Since solutions of BrAcT4 and of BrAcT3 undergo partial decomposition on evaporation to dryness, suitable procedures for the preparation of these hormones in solid form and for storage in solutions have been devised.


A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes. Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.


Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage. Large collections of random peptides can be expressed on the N-terminus of the pIII protein of filamentous phage and screened for binding to antibodies and other receptors. In our previous work with a monoclonal antibody (3E7) (Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382, 1990), we showed that a high proportion of the selected peptides had relatively low affinity (Kd's greater than 1 microM). Here we describe conditions for selective enrichment of phage expressing high affinity peptides. This is done by allowing the phage to interact with a low concentration of 3E7 Fab followed by extensive washing to allow dissociation of phage-bearing peptides with low affinity. These affinity selection conditions were applied to the pool of phage previously selected using a high concentration of IgG. A phage clone with the known high affinity ligand YGGFL (Kd 7.1 nM) and several other closely related peptides were isolated. The dissociation rate of 125I-3E7 Fab from several phage clones approximated that of phage expressing YGGFL. A good correlation was found between the dissociation rate of the peptides found on phage and the equilibrium binding constants of chemically synthesized peptides. The strategy of using a low concentration of receptor and extensive washing to select phage-bearing high affinity peptides, combined with assays to determine the specificity and relative affinity of peptides on isolated phage clones, should be generally applicable in using the peptides-on-phage system for discovery of high affinity receptor ligands.


Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils. S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (O2+) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The half-life (mercuric acid diazotization) of SNO-GSH in Hepes was greater than 60 min. The addition of resting PMN did not affect the T1/2 of SNO-GSH. PMN exposed to N-fMet-Leu-Phe (FMLP, 10(-7) M) reduced measurable SNO-GSH (15 microM) at 5 min (48 +/- 5.0% control, P less than 0.05). Incubation (5 min, 37 degrees C) of PMN with 10 microM tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 +/- 3 vs 78 +/- 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation.


Development of an avidin-biotin competitive inhibition assay and validation of its use for the quantitation of human intervertebral disc serine proteinase inhibitory proteins. A simple convenient method has been developed for the quantitation of serine proteinase inhibitors (SPIs) in tissue extracts. The method is based on the competitive binding to trypsin and chymotrypsin immobilized using glutaraldehyde on 96-well microtiter plate wells of native SPIs and a biotinylated secretory proteinase inhibitor (SLPI) standard. The bound SLPI standard was visualized using an avidin-alkaline phosphatase conjugate and inhibition curves were determined using absorbancy measurements at 405 nm. The standard assay had a range between 0.02 and 1 microgram SLPI/well and a lower detection limit of 20 ng SLPI/well; an improved microassay had a detection limit of 2 ng SLPI/well. Only active free inhibitor was detected in the assay since denatured and/or enzyme-inhibitor complexes did not bind to the plates. A range of SPI species was demonstrable in human bronchial mucus and intervertebral disc SPI samples using this technique. Quantitation of SPI levels in a number of intervertebral disc samples indicated that the SPIs were depleted in degenerate discs compared to nondegenerate discs (P less than 0.05, n = 12). Since the immobilized trypsin and chymotrypsin microplates used in this assay may be prepared in advance (and are stable at 4 degrees C for at least 1 month) the remaining two steps of the assay (the inhibition step and visualization) may be completed in 2-3 h; thus the assay is simple, convenient, and fast. All reagents (other than the biotinylated SLPI standard) are readily available commercially, and in principle the assay could be adapted to other systems provided defined biotinylated standards were available.


High-performance liquid chromatographic assays for free and phosphorylated derivatives of the creatine analogues beta-guanidopropionic acid and 1-carboxy-methyl-2-iminoimidazolidine (cyclocreatine). Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack of quantitative techniques to determine tissue content of these compounds. We describe the separation and quantitation of substituted guanidino compounds and their phosphorylated forms by high-performance liquid chromatography. First, a cation-exchange column is used to assay free creatine and its unphosphorylated analogues, and then phosphocreatine and its phosphorylated analogues as well as adenylate content (AMP, ADP, ATP) are assayed on an anion-exchange column. These methods have proven successful in measuring the chemical contents of these compounds in neutralized perchloric acid extracts of mammalian skeletal muscles. The sensitivity of this method ranges from 50 to 200 pmol, which is adequate to provide information from tissue extracts of 5- to 10-mg samples.


Sulfated proteoglycans of rabbit aorta: selective extraction and alternative method for glycosaminoglycan moiety analysis. Three different solutions containing urea, guanidine hydrochloride, or a detergent mixture were used to extract proteoglycan molecules (PG) metabolically labeled with 35S from normal rabbit aortic tissue. The size distribution of whole sulfated PG and the glycosaminoglycan (GAG) compositions in the three extracts were compared and found to be characteristically determined by the type of solution used for extraction. The spectrum of sulfated PG isolated by each solution was maintained at consecutive extractions of the tissue, even if this was used after another type of solution. The extracts obtained by using the urea- or guanidine-containing solutions contained similar, rather balanced populations of large and small PG, while the detergent-containing buffer was found to preferentially extract smaller, heparan sulfate-rich aortic PG. The selectivity of various extracting solutions could be exploited to obtain preparations enriched in certain types of sulfated PG. On the other hand, one could obtain a larger variety of 35S-labeled PG from the tissue by consecutively using two solutions with different capacities of extraction. The distribution of GAG moieties among PG populations, separated by size chromatography, was investigated by one of the commonly used methods and by a new method. The standard method is based on comparison of the chromatographic profiles of the extract before and after enzymatic digestions, requiring several chromatographic runs for a sample. In the alternative method proposed, the fractions obtained after a single chromatographic separation are adsorbed onto a support membrane. Processing of the whole membrane by GAG-specific, enzymatic treatments allows simultaneous assessment of GAG types in each fraction.(ABSTRACT TRUNCATED AT 250 WORDS)


Application of a novel apparatus, the quartz chemical analyzer, to the determination of endotoxin in blood. A novel apparatus called a quartz chemical analyzer (QCA) has been developed using a quartz crystal resonator. This apparatus measures sample viscosity changes based on resonant frequency changes of the quartz crystal. The apparatus was used to determine bacterial endotoxin concentrations by monitoring the gelation reaction of Limulus amebocyte lysate. The QCA determined endotoxin concentrations with good accuracy and reproducibility in the range of 0.001-3 EU/ml for endotoxin standard (JP XII). For endotoxin determination in human whole blood and plasma samples, the inhibitory reaction was eliminated by pretreatment of a fourfold dilution at 60 degrees C and incubation for 30 min. There are many advantages of the QCA method compared with the turbidimetric and chromogenic methods. For example, QCA can measure sample viscosity changes with high sensitivity and accuracy because QCA detects minor resonant frequency changes and the frequency data give a numerical value for easy quantitation. QCA can examine turbid samples, and the required quantities of samples and reagents are small, since the quartz crystal detects sample viscosity changes directly. The endotoxin determination time may be shortened by raising the reaction temperature, and QCA can detect other types of coagulation reactions.


Spectroscopic quantitation of organic isothiocyanates by cyclocondensation with vicinal dithiols. Organic isothiocyanates are widely distributed in plants and are responsible for a variety of beneficial and toxic biological effects. No direct and generic method for quantitating isothiocyanates has been described. Under mild conditions nearly all organic isothiocyanates (R-NCS) react quantitatively with an excess of vicinal dithiols to give rise to five-membered cyclic condensation products with release of the corresponding free amines (R-NH2). The products of the condensation of propyl-NCS with 1,2-ethanedithiol, 2,3-dimercaptopropanol, and 1,2-benzenedithiol have been isolated and identified as 1,3-dithiolane-2-thione, 4-hydroxymethyl-1,3-dithiolane-2-thione, and 1,3-benzodithiole-2-thione, respectively. Since 1,3-benzodithiole-2-thione (lambda max 365 nm and alpha m 23,000 M-1 cm-1) can be sensitively measured spectroscopically, the reaction of organic isothiocyanates with 1,2-benzenedithiol has been developed for analytical purposes. All aliphatic and aromatic isothiocyanates tested (except tert-butyl and other tertiary isothiocyanates) reacted quantitatively with an excess of 1,2-benzenedithiol. Thiocyanates, cyanates, isocyanates, cyanides, or related compounds did not interfere with this reaction under assay conditions. The method can be used to measure 1 nmol or less of pure isothiocyanates or isothiocyanates in crude mixtures. It can also be used to measure isothiocyanates in chromatographic fractions obtained from plant extracts and for the assay of the rate of cleavage of glucosinolates by myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1).


An electrophoresis-based assay for glycosyltransferase activity. Polyacrylamide gel electrophoresis (PAGE) and capillary zone electrophoresis (CZE) were used to measure the activity of glycosyltransferases. Acceptor molecules were prepared by reductive amination of the monopotassium 7-amino-1,3-naphthalenedisulfonic acid (AGA) Schiff base with sugars. The resulting sugar conjugates were purified by gradient PAGE and recovered using semidry electrotransfer into a positively charged nylon membrane. The beta(1----4)galactosyltransferase was shown, by PAGE analysis, to transfer a beta-galactosyl residue to the AGA conjugate of beta-D-GlcNAc-(1----4)-beta-D-GlcNAc-(1----4)-D-GlcNAc (compound 4). Similarly, alpha(1----2)fucosyltransferase isolated from porcine submaxillary glands was shown to transfer fucose from GDP-fucose to the AGA conjugate of beta-D-Gal-(1----4)-beta-D-GlcNAc-(1----6)-D-Gal (compound 5). This conjugate (compound 5) was also an acceptor for the alpha(1----3/4)fucosyltransferase partially purified from human milk. The latter reaction was followed by both gradient PAGE and CZE, having sensitivities of 200 pmol and 80 fmol, respectively.


High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity. High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.


Detection of receptor-ligand interactions using surface plasmon resonance: model studies employing the HIV-1 gp120/CD4 interaction. Surface plasmon resonance (SPR), a label-free, real time optical detection principle, has been investigated for its potential to detect and quantitate macromolecular ligand-ligate interactions. As model systems, the interactions of the HIV-1 envelope glycoprotein, gp120, and the monoclonal antibody L-71, with a soluble form of the T-cell receptor CD4 (sCD4), were investigated. In an effort to demonstrate potential analytical applications of this technology, operational characteristics of the SPR instrumentation (BIAcore, Pharmacia) including stability of the sensing surface and reproducibility in the measurement of such macromolecular interactions were investigated. In addition, the ability to detect and quantitate sCD4 directly from unfractionated cell culture supernatants, such as Streptomyces lividans, was investigated. The results demonstrate that SPR has potential in quantitating macromolecular interactions in both purified and crude samples and that the reproducibility in, and sensitivity of, such determinations is comparable to other techniques.


Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector. Surface plasmon resonance detectors, such as the BIAcore instrument produced by Pharmacia, show promise for the detection and quantitation of macromolecular interactions in a label-free mode. Such detectors rely on the covalent immobilization of one of the interacting species onto the sensing surface. To date, the only published chemistry for this purpose is reaction of primary amino-containing ligands with an N-hydroxysuccinimide (NHS) ester-activated surface. In an effort to increase the versatility of the BIAcore with respect to immobilizing ligands, we undertook an investigation of activation chemistries compatible with this system. Using readily available reagents, we demonstrated that the carboxylated dextran-coated sensing surface could be easily converted to functions other than NHS-esters, including amine-activated, hydrazine-activated, and sulfhydryl-activated surfaces. In addition, use was made of the streptavidin/biotin interaction to probe chemical modifications of the sensing surface, by employing specifically modified biotin derivatives.


High-performance liquid chromatographic separation with electrochemical detection of amino acids focusing on neurochemical application. Twenty-three amino acids and dipeptides, including compounds of neurochemical interest, are measured by high-performance liquid chromatography using electrochemical detection after precolumn derivatization with o-phthalaldehyde/beta-mercaptoethanol. The method uses a multistep polarity gradient system and the entire separation is performed in less than 23 min of analysis. The minimum detectable quantity was 0.66 pmol injected, corresponding to 50 nM concentration in the sample; the response was linear in the tested range of 1.33-1333 pmol (0.1-100 microM). Relative standard deviations ranged from 0.75 to 6.089% for area measurements (mean, 2.33) and from 0.209 to 0.779% for retention times (mean, 0.546). Examples of application of the method to analysis of biological samples such as rat brain homogenate and human cerebrospinal fluid are shown.


Same-day batch measurement of glycine betaine, carnitine, and other betaines in biological material. Glycine betaine, carnitine, carnitine esters, butyrobetaine, and proline betaine (stachydrine) concentrations in biological materials can be reliably measured in 100-microliters samples, with a detection limit below 1 mumol/liter. The procedure is suitable for batches of more than 30 specimens and it is possible to obtain a single result within 2 h. The betaines are extracted into an acetonitrile:methanol mixture, dried with anhydrous disodium hydrogen phosphate containing argentous oxide. The 4-bromophenacyl ester derivatives are formed using 4-bromophenacyl triflate as reagent, in the presence of solid magnesium oxide as base. The derivatives are separated by high-performance chromatography on a silica column, in a mixed partition and ion-exchange mode.


Analysis of individual-site binding data. Individual-site isotherms add experimental data which may allow for a more detailed definition of the parameters in a system with interacting binding sites. Individual-site isotherms accomplish the following: (A) In general, they define little more than the total or combined isotherm except to reveal the existence of different sites. (B) Under the limiting conditions of symmetrical interactions in two site systems they define: (1) the ratio of the unperturbed or intrinsic binding constants rather than their actual values, (2) the unperturbed shape of the total isotherm, that is, the shape of the total isotherm if there were no ligand dependent interactions between the sites, and (3) the perturbation of the shape of the total isotherm derived from interactions between the sites. (C) They do not define the nature of the interactions; that is, they do not resolve the free energies of the interactions between the sites. (D) When some assumptions about the nature of the interactions are made they may aid in defining some free energies of interaction between the sites.


A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: identification of the positions of carbohydrate-linked asparagine in recombinant alpha-amylase by treatment with peptide-N-glycosidase F in 18O-labeled water. Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of Asn-linked carbohydrates, was successfully applied to identification of N-glycosylation sites in a glycoprotein with the known or DNA-derived sequence (S. A. Carr and G. D. Roberts, 1986, Anal. Biochem. 157, 396-406). Here, we extended the method for easier identification of N-glycosylation sites in a glycoprotein even with unknown sequence. The glycoprotein is digested with peptide-N-glycosidase F in buffer containing 40 at% H2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their beta-carboxyl group during this digestion. The deglycosylated protein is further digested with proteolytic enzymes in an appropriate buffer prepared with normal water, and then peptides are separated on a reversed-phase column by HPLC. Peptides in which carbohydrate-linked Asn has been converted to Asp show a pair of signals ([M + 1]+ and [M + 3]+) in FAB mass spectra due to the partial incorporation of 18O into the beta-carboxyl groups of Asp residues, while the other peptides show normal isotopic ion distributions. Thus, both formally N-glycosylated peptides and, using collision-induced dissociation analysis, N-glycosylation sites can be identified. The application of the present method to the determination of N-glycosylation sites in a recombinant glycoprotein, Bacillus licheniformis alpha-amylase, is described.


A mass spectrometric method for measuring glycerol levels and enrichments in plasma using 13C and 2H stable isotopic tracers. The stable isotope tracer [1,1,2,3,3,-2H5]glycerol has been commonly used as a tracer to measure glycerol kinetics and lipolysis in vivo. The method for measuring samples using the trimethylsilyl derivative and electron impact gas chromatograph-mass spectrometry retains only three of the five deuteriums, resulting in the possibility of incorrectly identifying the whole glycerol tracer molecule. This reports preparation of glycerol as the heptafluorobutyrl derivative and measurement by negative ion chemical ionization gas chromatography-mass spectrometry to produce a derivative with an intense molecular ion that retains all five deuterium labels. Thus the heptafluorobutyrl derivative analyzed by negative ion mass spectrometry overcomes the problems associated with fragmentation and loss of the isotopic label. Glycerol concentration using a labeled internal standard can be determined in plasma with a precision of 3%. Nanomole amounts of glycerol can be analyzed for 13C or 2H enrichments with a precision of +/- 0.14 mol% excess isotope. This simple, sensitive method for measuring glycerol levels and stable isotopic enrichment in plasma uses a simple extraction procedure and requires a minimal volume of plasma (less than 300 microliters).


Analysis of reactive carbonyls in the expired air of transgenic mice. Methods for the determination of trace levels of volatile carbonyl compounds in air expired from mice were developed and validated. Tumor bearing transgenic mice or nontransgenic control mice were placed into a glass chamber through which air was passed continuously at 90 ml/min for 1 h. The effluent gas stream was bubbled into an aqueous cysteamine solution or an aqueous methylhydrazine solution. Formaldehyde, acetaldehyde, and acetone in expired air were derivatized to thiazolidine with cysteamine and malonaldehyde was derivatized to 1-methyl-2-pyrazole with methylhydrazine. The derivatized compounds were analyzed by capillary gas chromatography with flame photometric or nitrogen-phosphorous-specific detection. The lowest level quantitated was 4 micrograms/ml thiazolidine, equivalent to 1.35 micrograms/ml formaldehyde. Formaldehyde was recovered at a level of 1356 +/- 234 nmol/kg0.75 (mean +/- SD) from mice with tumors and 898 +/- 97 nmol/kg0.75 from mice without tumors, suggesting that tumor bearing transgenic mice expired significantly more formaldehyde than did tumor free controls. Amounts of expired acetaldehyde and acetone were not different among mice. Malonaldehyde was not detected in either group of mice.


Efficient precipitation and accurate quantitation of detergent-solubilized membrane proteins. The protein assay method of I. Polacheck and E. Cabib (Anal. Biochem. 117, 311-314, 1981) has been modified to provide a general method for quantitating protein samples in the presence of detergents. Dilute detergent-solubilized membrane proteins, by using ribonucleic acid as a carrier, have been efficiently precipitated here by trichloroacetic acid (TCA) in the presence of sodium dodecyl sulfate (SDS). Washing the pellets once with TCA solution has removed most of the reagents present in the original sample. The washed sample could then be quantitated by the Lowry method (O. J. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265-275, 1951). This procedure could be used to assay protein solutions of a concentration as low as 5 micrograms/ml in the presence of the following reagents: Triton X-100, Triton X-114, Tween 20, N-octylglucoside, deoxycholate, cholate, Thesit, octanoyl-N-methylglucamide, isotridecylpoly (ethyleneglycoether)n, Nonidet P-40, glucose, methyl-D-glucopyranoside, methyl-D-mannopyranoside, N-acetyl-glucosamine, Mn2+, Ca2+, Mg2+, and many buffer reagents. Proteins solubilized from porcine brain myelin sheath and synaptic plasma membranes were quantitated by amino acid analysis and by the TCA/SDS precipitation method described here. The resultant protein concentrations were almost identical. The results have suggested this TCA/SDS precipitation method to be useful for quantitating dilute protein samples containing high concentrations of detergents and other reagents commonly employed in studying membrane proteins.


Selective 5' modification of T7 RNA polymerase transcripts. We have developed two methods for selective 5' modification of RNAs generated by enzymatic synthesis using T7 RNA polymerase. The first method involves a two-step procedure. Transcription reactions are performed under standard conditions except that GTP is replaced by GTP gamma S. Since the polymerase initiates transcription with GTP, every transcript contains a 5' gamma-thiophosphate group which is modified with the thiol-specific reagent of choice (e.g., iodoacetyl dansyl derivative) in the second step. In an alternative method, transcription and modification reactions are carried out in a single step, using a mixture of dansylated GTP and GTP. Under the appropriate conditions, dansylated GTP effectively competes with GTP in the initiation reaction but does not substantially inhibit the elongation reaction. Yields of fluorescent 64-mer RNA ranging from 30 to 70% of the total transcription product have been obtained using these methods in combination with HPLC purification. This approach is amenable to large scale synthesis reactions and can be used to produce a wide variety of 5'-modified RNAs of virtually any size for structural or functional studies.


A polymerase chain reaction-based method for isolation of gene-specific sequences from the interferon-alpha gene cluster. The interferon-alpha gene is a gene family of over 20 distinct genes having 80-95% homology with one another at a nucleotide level. Because of the high homology in the gene cluster, the available interferon-alpha gene probes can hybridize to multiple bands of different size on Southern blot analysis of restricted human genomic DNA. We used the polymerase chain reaction with the primers synthesized from Alu repetitive sequence and the conserved sequences of the interferon-alpha gene cluster to generate specific probes for individual interferon-alpha genes. The amplification products were subcloned into a plasmid vector and analyzed by DNA sequencing and Southern blotting of the restricted human placental DNA. One clone, which derived from interferon-alpha 14 gene, produced a single 5.2-kb band in Southern blots of the HindIII-restricted human placental DNA. This stands in contrast to the 10 bands of different size that were detected with a cDNA for the interferon-alpha I' gene. Our results indicate that a polymerase chain reaction-based method can be used to isolate gene-specific sequences from the interferon-alpha gene cluster. Since a variety of human cancers has been found to have the complete or partial deletion of the interferon-alpha gene cluster, the gene-specific probe generated by this method may aid in determining the breakpoints in the vicinity of the gene cluster.


A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein. A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.


The use of streptavidin-biotinylglycans as a tool for characterization of oligosaccharide-binding specificity of lectin. A new rapid and sensitive method for characterizing lectin specificity using streptavidin-biotinylglycans as a tool is presented. This assay is analogous to enzyme immunoassay and takes advantage of the strong, irreversible adsorption of streptavidin to the wells of the chambers of titer plates. A series of streptavidin-biotinylglycans was first coated on a microtiter plate, and then one of six lectins, concanavalin A, wheat germ agglutinin, Phaseolus vulgaris (red kidney bean) erythro-agglutinin, Lens culinaris (lentil) agglutinin, Datura stramoniun agglutinin, or Sambucus nigra (elderberry bark) agglutinin coupled to horseradish peroxidase, was added. After incubation and thorough washing, only the lectin bound to a complementary glycan remained and could be detected and quantified by the peroxidase reaction. It was established that the lectins retained their oligosaccharide-binding specificities after coupling to the peroxidase, that the binding was inhibited by addition of the corresponding sugar inhibitors, and that the color intensity produced by the enzyme reaction is proportional to the amount of lectin-peroxidase bound to biotinylglycan complexed with streptavidin immobilized on the plate. As an example, it was found that the peroxidase-D. stramoniun agglutinin conjugate strongly bound biotinylglycans, GlcNAc3-Man5-R, GalGlcNAc3Man5-R, and GlcNAc3-4Man3-R (R = GlcNAc2-[6-(biotinamido)hexanoyl]-Asn). As little as 10 pmol/ml of lectin was detected. With the growing availability of biotinylglycans, the method should represent a reliable and simple procedure for screening lectin-oligosaccharide recognition qualitatively and quantitatively.