Prospective comparison of duplex scanning and descending venography in the assessment of venous insufficiency. A prospective study comparing duplex scanning and descending venography was applied to 143 venous segments in 25 extremities with moderate to severe manifestations of chronic venous insufficiency (class 2 or 3). Duplex scanning was performed with the patient in the 15 degree reverse Trendelenburg position, and descending venography with the patient in the 60 degrees semi-erect position; the Valsalva maneuver was used to elicit reflux in both tests. The duplex parameter of reflux duration greater than 0.5 second correlated with venographic reflux in 94 of 105 segments (sensitivity of 90%). Conversely, reflux time less than or equal to 0.5 second correlated with venographic competence in 32 of 38 segments (specificity of 84%). A total of 17 discrepancies were identified among the 143 total segments studied, for an accuracy of 88%. The largest proportion of discrepancies was identified in the group with venographic competence and reflux duration greater than 0.5 second and less than or equal to 2.0 seconds; this was designated a gray zone. Mean peak velocities were significantly higher in the reflux group when compared with the competence group in the profunda femoris vein (p = 0.047), greater saphenous vein (p less than 0.001), popliteal vein (p less than 0.001), and tibial vein (p = 0.005). We conclude that venographic reflux correlates best with duplex scan findings of reflux duration greater than 0.5 second. Duration of reflux greater than 0.5 second and less than or equal to 2.0 seconds, however, represents a gray zone and should be interpreted with caution since this could lead to over-reading of reflux disease, in which case verification of incompetence by descending venography may be indicated.


Physiologic similarities between extremities with varicose veins and with chronic venous insufficiency utilizing air plethysmography. Air plethysmography (APG) was used to measure maximal venous outflow rate (MVO), a test for proximal venous obstruction; venous volume; venous filling index, an estimate of valvular incompetence; ejection fraction (EF), a test of calf-muscle pump efficiency; and residual volume fraction (RVF), an estimate of ambulatory venous pressure. MVO was lower in patients with chronic venous insufficiency (CVI) than in those with varicose veins (VV), but the difference was small (p = 0.06). RVF was significantly greater in extremities with CVI when compared with those with VV (p less than 0.01). However, the degree of abnormality in venous volume, venous filling index, and EF was similar in CVI and VV extremities. In summary, although RVF tends to be higher in extremities with CVI when compared with those with VV, there is tremendous overlap between VV and CVI for each of the hemodynamic variables measured by APG. Therefore, the pathophysiology of CVI is likely to involve not only hemodynamic abnormalities but also other factors that have not yet been clearly identified.


Why does prophylaxis with external pneumatic compression for deep vein thrombosis fail? External pneumatic compression (EPC) devices are increasing in popularity for deep vein thrombosis (DVT) prophylaxis. Patients who have these devices applied postoperatively are assumed to have effective prophylaxis, although a number of extensive postoperative DVT complications have been observed. This study evaluates the proper application of EPC devices in patients in intensive care units and regular nursing floor units and assesses whether dedicated in-service instruction can improve proper use. In a prospective study of 138 patients with 2 or more risk factors for postoperative DVT, it was found that patients on routine nursing units had properly functioning EPC devices during 48% (306 of 636) of the visits compared with 78% (312 of 398) of the visits in the intensive care unit (ICU) (p less than 0.0001). Follow-up of patients transferred from an ICU to a regular nursing unit showed that functional application decreased from 82% (129 of 157) to 33% (40 of 122) (p less than 0.005). The compression sleeves were not applied in 84% of the nonfunctional devices and were properly in place but the pump nonfunctional in 16%. Unfortunately, dedicated in-service instruction did not improve the proper use of EPC. Although proper application of EPC is better in the ICU compared with regular nursing units, improper use is frequent and failure of DVT prophylaxis with EPC devices may be due to improper use, rather than failure of the method itself.


Operative management of greater saphenous thrombophlebitis involving the saphenofemoral junction. Forty-three consecutive patients with greater saphenous vein (GSV) thrombosis extending to the saphenofemoral junction (SFJ) were treated. Twenty-three patients had extension of thrombus into the common femoral vein (CFV). Twenty patients had thrombus extending to but not within the CFV. Symptoms, risk factors, and physical examination were not predictive of CFV thrombus extension. When compared with the operative record, duplex scans accurately located the extent of the thrombosis 100% of the time. Forty-one surgical procedures were performed. No patients had pulmonary emboli during the procedures. Thirty-seven patients were treated as outpatients or were discharged within 3 days of their surgical procedures. The two patients who did not undergo operative procedures in this series had complete occlusion of the CFV with extension into the external iliac vein. Thrombus within 3 cm of the SFJ is an indication for surgical intervention. Disconnection of the GSV from the CFV prevents extension of the thrombus, and a limited CFV thrombectomy can be performed when necessary. This is considerably more cost-effective than treatment with anticoagulation.


Are there angiographic predictors of success for vein bypass to the peroneal artery? In order to evaluate the effect of angiographic run-off upon peroneal artery autogenous vein bypass patency and subsequent limb salvage, 53 autogenous vein peroneal artery bypasses performed for ischemic tissue loss were reviewed. All preoperative angiograms were evaluated according to three separate angiographic scoring systems that previously had been designed to quantify the severity of runoff resistance. None of the three scoring systems predicted either early bypass occlusion and/or major amputation. The cumulative 18-month primary patency and 24-month secondary patency rates were 71% and 90%, respectively. The 24-month limb salvage rate was 81%. Initial and intermediate-term salvage of limbs with ischemic tissue loss can be achieved by peroneal artery bypass. Angiographic scoring systems were poor predictors of bypass failure or major amputation.


Clinical and hemodynamic results of bypass to isolated tibial artery segments for ischemic ulceration of the foot. Patients with ischemic ulceration of the foot and no continuous tibial or inframalleolar vessels acceptable for bypass are often treated with primary amputation. We have performed autogenous vein bypass to isolated tibial artery segments (ITAS) in nine patients with ischemic foot ulcers and no other outflow options. We reviewed the clinical and hemodynamic results of these bypasses to assess the efficacy of this approach. Hemodynamic comparisons of these ITAS bypasses were made to a concurrent series of 26 bypasses to intact tibial arteries and 24 inframalleolar artery bypasses assessed during routine follow-up. Eight of the bypasses originated from the above-knee popliteal artery and one from the profunda femoris artery. Recipient vessels were the anterior tibial (seven), peroneal (one), and posterior tibial (one) arteries. Although mean ankle brachial indices (ABI) increased significantly from 0.26 +/- 0.06 preoperatively to 0.75 +/- 0.04 postoperatively (p = 0.0015), ITAS bypass patients had lower mean postoperative ABIs than patients with bypasses to intact tibial (ABI = 0.98 +/- 0.03, p = 0.0001) or pedal arteries (ABI = 1.02 +/- 0.04, p = 0.0005). Similarly, duplex scan-derived peak systolic flow velocities of the ITAS bypasses (mean: 52.9 +/- 5.8 cm/sec) were lower than those of intact tibial artery bypasses (mean: 80.1 +/- 6.1 cm/sec, p = 0.02) but did not differ from those of pedal bypasses (mean: 59.5 +/- 3.5 cm/sec, p = 0.34). No ITAS bypass grafts have failed during a mean follow-up of 12.3 +/- 2.7 months. Although wound healing was prolonged (mean: 3.1 +/- 0.6 months), the wounds of eight of nine patients eventually healed, with three patients requiring minor amputations (one digital amputation and two transmetatarsal amputations). Although the hemodynamic results of ITAS bypass are inferior to those of more conventional bypasses, the early patency rates and successful healing of ischemic wounds confirm that it is a valid alternative in the threatened limb with no other outflow options.


Clinical results of decompressive dermotomy-fasciotomy. Seventy-three dermotomy-fasciotomies (DFs) were performed in 68 patients from 1986 to 1991. A database record was compiled on each patient. Variables included age, mode of injury, method of initial wound closure, and associated injuries. A multivariate stepwise logistic regression analysis was performed to determine which variables were associated with wound complications. Thirty-eight percent of patients who underwent DF developed wound complications. One hundred percent of those patients with postoperative arterial or graft thrombosis developed wound complications (p less than 0.01) as did 78% of those with thromboembolic disease (p less than 0.05). Conversely, only 5% of those who underwent closure of their DF wounds utilizing skin grafts developed wound complications (p less than 0.01) as compared with 51% of those who underwent secondary or primary closure only. Subsequent analysis of the remaining patients, excluding those with severe soft tissue injury, showed an association between location of DF (upper versus lower extremity) and the development of wound complications that approached statistical significance (p less than 0.06). DF is frequently necessary in the treatment of patients with compartment syndrome but is associated with significant morbidity. This study suggests that closure of DF wounds utilizing skin graft allows for continued osteofascial decompression while concomitantly minimizing invasive sepsis.


Management of infected lower extremity autologous vein grafts by selective graft preservation. Between 1975 and 1991, we treated 16 patients with infected lower extremity autologous vein grafts performed for limb salvage by complete graft preservation. Traditional treatment of these infections includes immediate graft excision and complex revascularization procedures to prevent limb loss. The infection involved an intact anastomosis in 12 patients or the body of a patent graft in 4 patients. None of the patients was systemically septic. All patients were treated with appropriate intravenous antibiotics. Six patients were treated by placement of autologous tissue on the exposed graft (4 rotational muscle flaps, 2 skin grafts), and 10 were treated with antibiotic-soaked dressing changes and repeated operative débridements to achieve delayed secondary wound healing. This treatment resulted in a 19% (3 of 16) mortality rate and an 8% (1 of 13) amputation rate in survivors. Of the six patients managed by autologous tissue placement onto the infected graft, five patients had wounds that healed without complications, and one died of a myocardial infarction. Of the 10 patients treated by delayed secondary wound healing, 2 developed anastomotic hemorrhage, which resulted in death in 1 patient and above-knee amputation in the other, 1 died of a myocardial infarction, 1 developed graft thrombosis, and 6 had wounds that healed. Placement of autologous tissue to cover an exposed, infected patent vein graft with intact anastomoses may prevent graft dessication, disruption, and thrombosis, which renders graft preservation an easier, safer method of treatment compared with routine graft excision.


Donor iliac angioplasty and crossover femorofemoral bypass. We reviewed our experience with 99 patients who had 111 femorofemoral bypass grafts placed over a 10-year period. Mean follow-up was 36 +/- 28 months (range: 1 to 120 months). Bypass alone was performed in 89 cases (group 1). Preoperative donor iliac angioplasty was utilized in 22 cases (group 2). Overall graft failure was 21 of 89 in group 1 and 2 of 22 in group 2 (difference was not significant by chi 2: p greater than 0.05). Clinical success as calculated by life-table analysis was 95%, 83%, 75%, and 67% at 1, 3, 5, and 7 years, respectively, for group 1. Clinical success was 100% and 91% at 1 and 3 years, respectively, and 91% at 42 months for group 2. The success rates were not different for the two groups when analyzed by the log-rank test at 42 months (p greater than 0.30). We conclude that donor iliac angioplasty and femorofemoral bypass is an excellent option for patients with severe occlusive disease of one iliac artery and contralateral disease amenable to angioplasty.


Management of small artery vascular trauma. This 11-year retrospective study reviewed 99 arterial injuries distal to the brachial bifurcation or popliteal trifurcation in 89 extremities in 88 patients. Associated injuries occurred in 78 of 88 (89%) patients, including 10 fractures or dislocations, 66 nerve injuries, and 59 single or multiple tendon injuries. Fasciotomy was performed in 9 upper extremities and 11 lower extremities (23% of patients). The selection of operative treatment by arterial repair or ligation was by surgeon choice (52% repair and 48% ligation). Postoperative patency was found in 45 of 47 (96%) repaired arteries. In cases of isolated single arterial injuries (10), there were excellent results, and there was no difference in the results between repair and ligation. In cases of nonisolated single arterial injuries (69), there were 46% and 36% nonvascular complications in the repaired and ligated groups, respectively. In 10 patients with nonisolated multiple arterial injuries in the same extremity, the results of repair of one artery with ligation of the other artery versus repair of both arteries were identical, and there were no vascular complications. Operative exploration was the key to complete evaluation of vascular and neuro/musculoskeletal injuries. The data suggest that one functional artery distal to the elbow or knee is sufficient for limb viability and vascular function (follow-up range: 0 to 110 months; mean: 12 months). Nerve injury was the single most important factor of extremity injury in terms of the degree of functional loss.


Lower-extremity amputation with immediate postoperative prosthetic placement. To study the efficacy of an immediate postoperative prosthesis (IPOP) program, a retrospective review of 167 major lower-extremity amputations was performed. Patient enrollment in the IPOP program was based on the individual's potential for rehabilitation and participation in an aggressive postoperative physical therapy regimen, as determined by the surgeon, prosthetist, physical therapist, and social worker. Indications for amputation were intractable infection and/or severe unreconstructable arterial insufficiency. Sixty-five patients underwent 69 amputations with IPOP (59 below knee; 10 above knee). Successful program completion was defined as independent ambulation and occurred in 86% of those patients enrolled. The average interval from amputation to ambulation was 15.2 days for the below-knee amputees and 9.3 days for the above-knee amputees. Failure to complete the program occurred in 14% of patients and was due to noncompliance, stump infection, stump trauma, and death. The results of this review support the use of IPOP after major lower-extremity amputation.


Gastric antroplasty for the treatment of delayed gastric emptying and gastroesophageal reflux in children. Operative correction of symptomatic gastroesophageal reflux (GER) was undertaken in 530 children during a 21-year period. Gastroesophageal fundoplication (GEF) alone was performed in 415 children; 83 had simultaneous GEF and pyloroplasty; 13 had pyloroplasty alone; and 19 had GEF with later pyloroplasty. Of the last 355 children with reflux, 110 (31%) had pyloroplasty. Pyloroplasty was performed for persistent symptoms despite medical therapy when more than 60% of the isotope meal was retained in the stomach at 90 minutes. Children with central nervous system disorders and GER often had delayed gastric emptying (DGE). A modified Heineke-Mikulicz pyloroplasty was used for the first 59 children; the last 56 patients had a more simplified antroplasty with a 2.5- to 3.5-cm vertical incision through the antral muscularis down to the duodenum without mucosal incision; the muscularis was reapproximated in a transverse direction with sutures. With a mean follow-up of 5.8 years, only three patients experienced mild transient dumping. None had pyloroplasty leak or clinical evidence of alkaline reflux. Antroplasty is a helpful, simple adjunct to GEF with low morbidity in children with GER and DGE.


Nissen fundoplication in the treatment of children with familial dysautonomia. Thirty-four children with familial dysautonomia (FD) underwent Nissen fundoplication and gastrostomy. The indications for operation were persistent cyclic vomiting that resulted in repeated aspiration pneumonia (94% of the patients), chronic dehydration (82%), failure to thrive (97%), and frequent hospitalizations (76%). There was no operative or early postoperative mortality. Long-term follow-up for up to 12 years was available. Eight patients died during this period from 7 months to 7.5 years postoperatively. In 5 patients (15% of the operated patients), the fundoplication ceased to function 16 months to 5 years postoperatively, which was attributed mainly to repeated severe dysautonomic crises with vigorous retching. Vomiting ceased in 85% of the symptomatic patients; pulmonary deterioration was halted, and the frequency of aspiration pneumonia was reduced in 68%; nutritional improvement was seen in 44%; the hydration status improved in 88%; and the frequency of hospital admissions decreased in 74%. These long-term findings resulted in a significant improvement in the quality of life for the majority of the patients. The absence of operative mortality and the low postoperative morbidity, together with the long-term beneficial results of this surgical procedure, should encourage early surgical intervention in selected FD patients.


Further evidence against the routine use of parathyroid ultrasonography prior to initial neck exploration for hyperparathyroidism. The role of preoperative localization tests in patients undergoing initial neck exploration for hyperparathyroidism (HPT) is controversial. The use of parathyroid ultrasonography (US) in 46 patients (7 men, 39 women; mean age: 60 years) who underwent initial neck exploration for hypercalcemia and who had the diagnosis of HPT confirmed at surgery is reported. At surgery, a single adenoma was found in 40 patients and multiple hyperplastic glands in 6 patients. Of the 40 adenomas, only 22 (58%) were localized to the correct side by the preoperative US, and only 5 of 18 hyperplastic glands (28%) were correctly localized. The rates of false-positive and false-negative results were 10% and 46%, respectively. The sensitivity of parathyroid US was 54%, the specificity 90%, and the accuracy 70%. The low sensitivity and accuracy and the high rate of false-negative test results that were observed suggest that there is no role for the routine use of parathyroid US in patients undergoing initial neck exploration for HPT.


Lipocortin-present perforating and lipocortin-absent nonperforating Crohn's disease. To investigate whether Crohn's disease has two different clinical forms, a relatively aggressive perforating type and a more indolent nonperforating type, we compared the concentrations of lipocortin and prostaglandin E2 (PGE2) in the inflamed mucosa of 12 patients with strictly controlled Crohn's disease with those found in histologically normal mucosa of control subjects. The inflamed mucosa obtained from eight patients with nonperforating Crohn's disease did not react with antilipocortin antibody on immune blot analysis. In contrast, the inflamed mucosa from four patients with perforating Crohn's disease, as well as that obtained from histologically normal controls, contained lipocortins. In addition, higher concentrations of intramucosal ileal and colonic PGE2 were found in patients with nonperforating Crohn's disease (902 +/- 454 pg/wet weight [WW] mg and 1,268 +/- 567 pg/WW mg, respectively) compared with normal controls (90.2 +/- 43.1 pg/WW mg and 173 +/- 76.5 pg/WW mg, respectively) (p less than 0.01). The difference in intramucosal ileal and colonic PGE2 levels between patients with perforating Crohn's disease (109.6 +/- 16.7 pg/WW mg and 252 +/- 34.4 pg/WW mg, respectively) and normal controls was not significant. These findings indicate that there may be two distinct patterns of Crohn's disease that differ in the amount of lipocortin present in the intestinal mucosa.


Long-term follow-up of partial splenectomy in Gaucher's disease. Seven children with Gaucher's disease who underwent partial splenectomy were followed for 7 to 8 years. None of the children had systemic sepsis or symptoms related to liver enlargement. Bone crisis occurred in only two children who had experienced bone crisis prior to partial splenectomy. This contrasts with the development of bone crises in five of six children who underwent total splenectomy at a similar age and who had previously been free of bone symptoms. Partial splenectomy should be regarded as a temporary solution in the treatment of hypersplenism and the mechanical compression related to the huge spleen. In 71% of the patients, massive enlargement and a severe to moderate degree of pancytopenia occurred again after 3 to 8 years; total splenectomy was required in three patients.


Collagen synthetic capacity throughout the uninjured and anastomosed intestinal wall. The capacity to synthesize collagen was measured throughout the intestinal tract of the rat, using an in vitro technique. In addition, the effect of anastomotic construction in either the ileum or the colon on collagen synthetic capacity at specific distant locations both 1 and 4 days after operation was investigated. Collagen synthetic capacity is relatively uniform throughout the large bowel. However, in the small bowel in which synthesis is lower than in the large bowel, synthesis is significantly higher in the most proximal and distal segments than in the intermediate tissue. Anastomotic construction in either part of the bowel strongly increases collagen synthetic capacity at the immediate wound site. The synthetic response is not restricted to the anastomotic site but appears to be more generalized and is, in the small bowel, to some extent specific for collagen since the collagen synthetic capacity is increased far more than the capacity to produce noncollagenous protein. Anastomotic construction in the colon has only minor, although sometimes significant, effects on collagen synthetic capacity in the ileum, and vice versa.


Samuel Preston Moore: Surgeon-General of the Confederacy. Samuel Preston Moore was trained as a military surgeon in the US Army but resigned his commission and was appointed Surgeon-General of the Confederate States Army Medical Department at the beginning of the American Civil War. He reformed the mediocre medical corps by raising recruiting standards and improving treatment protocols and by placing the most capable surgeons in positions of authority. He improved the ambulance corps and directed the construction of many new hospitals for Confederate casualties. He was directly responsible for the barracks hospital design, which is still used today. He established the Confederate States Medical and Surgical Journal and directed a successful effort to develop substitutes for scarce pharmaceuticals from the indigenous flora of the South. He founded the Association of Army and Navy Surgeons of the Confederate States of America. With skill and dedication, Dr. Moore transformed the medical corps into one of the most effective departments of the Confederate military and was responsible for saving thousands of lives on the battlefield.


Abdominoinguinal incision in resection of pelvic tumors with lateral fixation. Large tumors in the area of the iliac fossa, external iliac vessels, wall of the lesser pelvis, and pubic bone are often considered unresectable through conventional abdominal incisions due to the lack of exposure, or they are managed with hindquarter amputation. The abdominoinguinal incision provides the necessary exposure for en-bloc resection of many of these tumors. It involves a lower midline incision that is extended transversely to the mid-inguinal point on the side of involvement and then vertically, over the course of the femoral vessels, which are exposed. The rectus abdominis is divided off the pubic crest and the inguinal ligament off the pubic tubercle, and the inferior epigastric vessels are ligated and divided at their origin. This allows in-continuity exposure of the lower abdomen, pelvis, and the groin in one field and dramatically improves the exposure.


Utility of an oral examination in a surgical clerkship. To evaluate the utility of the oral examination in a surgical clerkship, we designed a prospective and randomized study to relate the subjective impressions of experienced examiners with an objective measure of cognitive knowledge. The examiners were asked to score the student's performance as honors, high satisfactory, satisfactory, or unsatisfactory, according to their subjective impression of the student's ability. Student performance was grouped according to oral examination performance. The cognitive performance in the honors group was significantly better than that of the other groups (Student's t-test, p = 0.05). There was a significant difference in cognitive performance for oral examination groups throughout the rotations (analysis of variance, p = 0.000; Kruskal Wallis, p = 0.05). The oral examination is useful to identify a high level of cognitive achievement but cannot discriminate between groups of median to low competence. It should be used for educational feedback, career counseling, residency recommendations, and professional development.


Composite resident work week.

Composite resident work week. During a 4-week period, all residents in the Wright State University School of Medicine Department of Surgery residency program were required to keep a log of their daily activities. Times were collected for the following resident activities: patient rounds, work-ups, laboratory data review, consults, meetings with attending physicians or patient families, phone calls, teaching conferences, medical student teaching, library time, clinics, attendance at codes, emergency department visits, on-call time, medical records committee meetings, and time scrubbed. The composite average weekly hours by resident year were 83.4 hours [postgraduate year (PGY)-1], 78.4 hours (PGY-2), 60.2 hours (PGY-3), 79.6 hours (PGY-4), and 80.9 hours (PGY-5). More than 50% of the resident work week cards were for less than 80 hours. Three activities (patient rounds, time scrubbed, and on-call) accounted for the majority of the residents' hours, but the average week, deleting educational and administrative hours, was less than 80 hours for all residents.


Intestinal obstruction complicating pregnancy. Intestinal obstruction is a rare but serious complication of pregnancy with significant maternal and fetal mortality. The reported incidence of intestinal obstruction complicating pregnancy varies widely, from 1 in 66,431 to 1 in 1,500 deliveries. A retrospective review of 66 cases of intestinal obstruction complicating pregnancy and the puerperium, including 2 cases from our institution, revealed that the most common causes of mechanical obstruction were adhesions (58%), volvulus (24%), and intussusception (5%). Seventy-seven percent of the patients with obstruction due to adhesions had undergone previous abdominal or pelvic surgery. Presenting symptoms and signs were similar to those of the nonpregnant patient; abdominal pain was present in 98% of patients, vomiting in 82%, and tenderness to palpation in 71%. In 82% of patients, obstruction was evident on radiographic evaluation. Prompt management of obstruction is essential; the median length of time from admission to laparotomy in the 66 patients was 48 hours. Bowel strangulation requiring resection was present in 23% of patients. Thirty-eight percent of patients completed term pregnancies after operative resolution of obstruction; total maternal mortality was 6%, and total fetal mortality 26%. Thus, both mother and fetus are at risk when intestinal obstruction complicates pregnancy. Clinical suspicion of the presence of obstruction and aggressive intervention are required to decrease the morbidity and mortality of this rare complication of pregnancy.


Molecular mechanisms of tumor formation. DNA is the fundamental guide for the cell's processes. The alterations in DNA that can lead to abnormal or absent proteins and the role of chromosomal alterations in changing the function of the DNA are reviewed. The role of viruses in derailing a cell's normal functions, the major mechanisms of oncogene action, and tumor suppressor genes are also discussed.


The distribution of Merkel cells in human fetal and adult skin. Merkel cells (MCs) have been a subject of investigation in human and animal studies for over a century, but their origin, function(s), and exact distribution in human skin remain largely unknown. The objectives of the present study were to quantify these cutaneous neuroendocrine cells in fetal and adult human skin, using an immunohistochemical marker (neuron-specific enolase) and morphometric methods. Our results indicate that, in postnatal life, MC are sparsely distributed along the undersurface of epidermal and occasionally adnexal epithelium in an approximate range of 0.1-1.0 MCs per centimeter of basal zone. They are most numerous on volar skin and least on genital skin, and on the face they have a primarily perifollicular orientation. Fetal samples have shown that MCs appear between the 15th and 18th week of gestation and are most abundant on volar skin. They appear to arise within the epidermis, and are initially numerous and later diminish with increasing gestational age. Their predominance in intrauterine life suggests a functional role in growth and development.


Dermatofibroma with osteoclast-like giant cells. Dermatofibroma (DF), or cutaneous fibrous histiocytoma, is a common cutaneous tumor with many variants that may arise from alterations in the morphology and composition of its various components. One type that has not received much attention is DF with osteoclast-like giant cells. Two cases of this rare tumor are described. The importance of this tumor lies in the possible histologic confusion with a variety of benign and malignant neoplasms, including giant cell tumor of tendon sheath, giant cell tumor of bone, and giant cell reparative granuloma.


Evolution of B-cell lymphoma from pseudolymphoma. A multidisciplinary approach using histology, immunohistochemistry, and Southern blot analysis. Cutaneous B-cell lymphomas are rare neoplasms that can present as lesions involving solely the skin or develop in association with a systemic lymphoma. Histologically they are often difficult to differentiate from pseudolymphomas, and the use of immunohistochemistry may be necessary to correctly classify them. We report a study of multiple skin lesions in a patient who initially presented with multiple pseudolymphomas, apparently associated with an immune response to the dye of his tattoos. Over a period of 4 years his skin lesions evolved from histologically benign and immunologically polyclonal pseudolymphomas to a histologically malignant and immunologically monoclonal B-cell large cell lymphoma. Genotypic analysis with a probe for the heavy-chain immunoglobulin gene demonstrated the presence of clonal B-cell populations in all of the pseudolymphoma biopsy samples as well as in the subsequent lymphoma tissue samples, with a pattern of clonal bands suggestive of evolution of the B-cell clones. These findings suggest that the development of B-cell lymphoma in this patient was related to a persistent abnormal immune response to the chronic antigenic stimulus of the dye of the tattoo. The presence of clonal B-cell populations in pseudolymphoma by Southern blot analysis may be useful in predicting those patients who will subsequently develop overt lymphoma.


Intravascular spread of keratoacanthoma. An alarming but benign phenomenon. We report a patient with a keratoacanthoma of the scalp in which there was invasion of several medium-sized vessels by the tumour at a distance from the main lesion. A marked inflammatory response within the invaded vessels as well as a benign clinical course do not suggest that this phenomenon represents malignant transformation.


Multicentric reticulohistiocytosis. Immunohistological and ultrastructural study: a pathology of dendritic cell lineage. A case of multicentric reticulohistiocytosis is presented with extensive immunohistochemical study of the infiltrate in both paraffin and cryostat sections. Factor XIIIa dermal dendrocyte marker was demonstrated in the cytoplasm of histiocytes, which has not been reported previously in this disease. In addition, the S100 protein stained positive. This immunophenotyping study suggests a dermal dendrocyte lineage with an unusual expression of S100 protein.


Auricular granuloma annulare. A consequence of trauma? Localized granuloma annulare (GA) is a cutaneous disorder characterized by the formation of palisading necrobiotic granulomas. Although trauma and vasculitis have been considered, the etiology of most cases remains obscure and the disease is generally considered to be idiopathic. The condition may occur at any age and at any site, but usually involves the hands or feet of children or young adults, predominantly affecting females. We present two cases occurring as nodules involving the external ears of males. One of these showed a bilateral distribution and was clearly related to trauma.


Naming acquired melanocytic nevi. Common and dysplastic, normal and atypical, or Unna, Miescher, Spitz, and Clark? Acquired melanocytic nevi are among the commonest neoplasms in man, yet classification of them has proven elusive and elucidation unsatisfactory. From "active junctional nevus" to "dysplastic nevus," the brains of clinicians and histopathologists have been boggled and the psyches and skin of patients have been served badly. This essay advocates eponymic designations for acquired melanocytic nevi, all of which are common, as the surest route to comprehensive classification of them.


Regurgitation and oesophageal rupture with cricoid pressure: a cadaver study. The efficacy of cricoid pressure was studied in 10 adult cadavers. The oesophageal pressure that would result in regurgitation during measured values of cricoid pressure was determined. Oesophageal pressure, recorded by a 2 mm diameter oesophageal tube, was increased by oesophageal distension with saline, and incremental levels of cricoid force, 20, 30 and 40 Newtons, were applied with a cricoid yoke. With each 10 Newton increment of cricoid force there was a significant rise in the oesophageal pressure required to provoke regurgitation (p < 0.01). Thirty Newtons of cricoid force prevented regurgitation of saline in all cadavers with oesophageal pressures of up to 40 mmHg. Rupture of the oesophagus occurred in three cadavers: one at 30 and two at 40 Newtons of cricoid force, but there was no rupture at 20 Newtons of cricoid force. In the other seven cadavers oesophageal pressures were also studied with a 4.6 mm diameter (14 FG) oesophageal tube, which did not reduce the efficacy of cricoid pressure in preventing regurgitation.


Thoracic electrical bioimpedance measurement of cardiac output and cardiovascular responses to the induction of anaesthesia and to laryngoscopy and intubation. Noninvasive methods of determining cardiac output (by thoracic electrical bioimpedance) and arterial pressure (by intermittent oscillometry) were used to record minute-by-minute changes in heart rate, mean arterial pressure, stroke volume, cardiac output and systemic vascular resistance following induction of general anaesthesia and laryngoscopy and intubation in 60 healthy female patients who were either unpremedicated, or premedicated with temazepam or papaveretum-hyoscine. Anaesthesia was induced with a sleep dose (3-5 mg.kg-1) of thiopentone and maintained with 70% nitrous oxide in oxygen with 0.5-1% enflurane. Tracheal intubation was facilitated by administration of vecuronium 0.1 mg.kg-1. Mean arterial pressure and cardiac output decreased maximally 5 min after induction in all premedication groups by mean estimates of 21-25% and 14-22% respectively. Heart rate increased initially one minute after induction, but decreased to less than the baseline value 5 min after induction. Systemic vascular resistance was unchanged. The stimulus of laryngoscopy and tracheal intubation was accompanied by a significant pressor response and tachycardia one minute after intubation (with mean increases in mean arterial pressure and heart rate of 29-34% and 22-33% respectively). The increase in mean arterial pressure was secondary to an increase in systemic vascular resistance (36-57%), and was accompanied by a decrease in stroke volume (-25 to -31%). These changes were significant in all three groups. Cardiac output decreased only in unpremedicated patients. There were wide variations in the different haemodynamic indices.(ABSTRACT TRUNCATED AT 250 WORDS)


Implicit memory tested in children during inhalation anaesthesia. Memory for stimuli presented during inhalation anaesthesia was tested in 80 children undergoing eye surgery. Two groups were exposed, in a random double-blind study, to repeated neutral phrases including either the colour orange or green. A postoperative colouring task was used as a test of implicit memory to detect any preference for the colour named under anaesthesia. No colour preference attributable to implicit memory could be demonstrated. One patient had a distinct preference for the named colour. No patient remembered any intra-operative events.


Catecholamine response to laryngoscopy and intubation. The influence of three different drug combinations commonly used for induction of anaesthesia. The haemodynamic response and changes in plasma catecholamine concentrations associated with laryngoscopy and tracheal intubation were compared during anaesthesia employing three strictly standardised techniques with commonly used drug combinations. Thirty-six patients were investigated consecutively resulting in 12 patients in each of three study groups. Anaesthesia was induced with thiopentone 5 mg.kg-1 (group 1), fentanyl 6 micrograms.kg-1 with thiopentone 5 mg.kg-1 (group 2), or midazolam 0.2 mg.kg-1 with fentanyl 6 micrograms.kg-1 (group 3). Undesirable changes in haemodynamic effects and an elevation of plasma catecholamine concentrations during laryngoscopy and intubation occurred in group 1. Heart rate and mean arterial pressure increased significantly (34% and 23% respectively). Noradrenaline concentration increased by a maximum of 147%. The addition of fentanyl (groups 2 and 3) attenuated the adverse haemodynamic response and elevation of plasma catecholamine concentrations; heart rate and mean arterial pressure did not differ from pre-intubation values and plasma catecholamine concentrations decreased steadily. Substitution of thiopentone by midazolam in combination with fentanyl abolished the adverse haemodynamic response and modified the increase in plasma catecholamine concentrations. 'High-dose' opioid anaesthesia is not necessary to produce optimal conditions during laryngoscopy and intubation.


Opioid sensitivity of chronic pain: a patient-controlled analgesia method. Twenty-two patients with chronic pain of malignant or nonmalignant origin were given intravenous morphine by patient-controlled analgesia. A prestudy judgment was made from the characteristics of the pain as to whether it was nociceptive or neuropathic. Analgesic efficacy was assessed by a nurse-observer; adverse events were noted and plasma morphine and metabolitie concentrations measured. Three categories of opioid response were distinguished. Good responders obtained > 70 mm relief on the visual analogue scale, with minimal or manageable adverse events. Moderate responders obtained < 70 but > 30 mm relief with more problematic adverse events, and poor responders had < 30 mm relief with troublesome adverse events. This method for the study of opioid sensitivity allowed a wide dosage range to be studied. The simultaneous analgesic and adverse event measurements showed that the spectrum of observed response was wide, and response category could be judged for the majority by 4 h. In those with poor or moderate response, adverse event severity limited further dose increment. The relationship between pain characteristics and response showed that some pains judged to be neuropathic had a good response to opioid (5/13), and some pains judged to be nociceptive did not (5/14). The study suggests that the pattern of response is not as black and white as the prediction of good response from nociceptive pain and poor from neuropathic pain would suggest, although nociceptive pain was more likely than neuropathic pain to show a good response. For the moderate responders opioid titration may, in the absence of other effective treatments, be useful, but the analgesic endpoint may not be totally satisfactory. The method provides an operational definition of opioid sensitivity.


Isoflurane and propofol for long-term sedation in the intensive care unit. A crossover study. Propofol and isoflurane have been reported recently to offer better sedation than alternative agents in patients who require long-term ventilation in the Intensive Care Unit. This is the first report of a direct comparison between propofol and isoflurane. Twenty-four patients predicted to require artificial ventilation for at least 48 h were entered into a randomised crossover study to monitor sedation quality and time to recovery from sedation. There were no significant differences between the two agents in either end-point, with over 95% optimal sedation achieved by the use of each drug. Few adverse events were noted. Technological advances in the administration of volatile agents as long-term sedatives in the Intensive Care Unit may facilitate their more widespread use.


The Riyadh Intensive Care Program applied to a mortality analysis of a teaching hospital intensive care unit. A computerised system of prediction of death using the Riyadh Intensive Care Program was applied retrospectively over a 17-month period to data collected prospectively on 1155 patients admitted to our intensive care unit. Variables which enable organ failure scores to be generated were recorded daily to make these predictions. Consultant medical opinion predicted that outcome was hopeless in 55% (115/209) of the patients who died. The predictive power of the computer demonstrated a sensitivity of 14.8% and a specificity of 99.8%. It is possible that the occurrence of three false predictions of death in the latter part of the series may have been related to a change in our antibiotic policy. We would be unhappy to recommend the general use of a computerised program for prediction of death without careful explanation of its significance and dangers.


Acute isoniazid poisoning.

Acute isoniazid poisoning. We present two cases of acute isoniazid poisoning in patients who ingested 7.5 g and 5.0 g of isoniazid respectively, with the intention of committing suicide. Both were admitted unconscious, with ventilatory insufficiency and convulsions. Hepatic dysfunction and peripheral neuropathy were notable complications.


Does hypothermia protect against the development of hepatitis in paracetamol overdose? A 24-year-old female presented in hospital following self-poisoning with a dose of greater than 30 g of paracetamol (acetaminophen), taken both as co-proxamol (dextropropoxyphene and paracetamol) and paracetamol. She arrived in hospital more than 18 h after ingestion of the drug. On admission, she was profoundly hypothermic, with a rectal temperature of 19 degrees C. Her paracetamol level was 943 mumol.l-1 which, when related to the time of ingestion, implied a very high risk of hepatocellular damage as well as fulminant liver failure, even if she was treated with the antioxidant n-acetylcysteine. The patient's condition was stabilised by initial resuscitation with fluids, vasoactive drugs, and active rewarming. N-acetylcysteine therapy was begun promptly. This patient's liver function tests remained entirely normal in spite of the delay in presentation and she made a rapid and complete recovery. This remarkable clinical course indicates a possible role for therapeutically induced hypothermia in the management of severe paracetamol overdose, particularly in the group of patients who seek medical attention some hours after ingestion of the drug and who therefore remain at high risk, despite treatment with n-acetylcysteine.


Managing the airway in cervical spine injury. A review of the Advanced Trauma Life Support protocol. The American College of Surgeons' Advanced Trauma Life Support protocol for managing the airway in patients with a cervical spine injury is reviewed. The relative risks and benefits of oral and nasotracheal intubation are discussed and the potential hazards of alternative methods of airway control are considered with particular respect to British practice.


The pre-operative anaesthetic visit. Its value to the patient and the anaesthetist. One hundred and thirty-two patients staying in hospital more than 24 h were visited pre- and postoperatively. Patients were asked a standard set of questions, and 39% could not remember accurately what they were asked. This has important medicolegal implications. In 15% of patients, information that significantly altered subsequent anaesthetic management was discovered, but in less than 3% would ignorance of the patient's condition have required postponement of the surgery. We conclude that the major reason for a pre-operative visit by an anaesthetist is that patients appreciate it, rather than it being medically necessary.


Chronic pain management training for senior registrars in anaesthesia. In 1985 and 1990 postal questionnaires were sent to anaesthetic senior registrars in training in the United Kingdom to determine the extent of higher specialist training in chronic pain management. There were wide variations in training and experience amongst senior registrars. Overall there was little change between 1985 and 1990. In particular the number of anaesthetic senior registrars who felt equipped to undertake a consultant post with an interest in chronic pain management had not increased.


[Perioperative diagnosis of acute myocardial ischemia]. The prevalence of coronary artery disease substantially affects both cardiac and noncardiac surgery. Assuming that biometric data reported from North America are representative for Germany, the following incidences can be estimated: around 1 million out of 8 million patients operated upon each year will suffer from coronary artery disease, and 15,000 of these patients will have a perioperative myocardial infarction. Since a close relationship has been shown between pre-, intra-, and postoperative myocardial ischaemia and postoperative cardiac morbidity and mortality, early diagnosis and therapy of acute perioperative myocardial ischaemia is warranted. The purpose of this review is to weigh critically the various methods for diagnosis of myocardial ischaemia in view of their practicability and cost/benefit relationship in the perioperative setting. The symptoms of angina pectoris are unreliable in the perioperative period, since patients are premedicated preoperatively, without symptoms during anaesthesia, and usually receive analgesics postoperatively. Intraoperative detection of myocardial ischaemia focuses on standard electrocardiography (ECG) with on-line registration of the ST-segment in two leads (usually leads II and V5) and automatic analysis of ST-segment deviation, achieving a sensitivity of 80% in the detection of myocardial ischaemia. Measurement of regional wall motion abnormalities with trans-esophageal echocardiography (TEE) is a more sensitive method of myocardial ischaemia detection compared to ECG. However, several reasons preclude the broader application of this method in the perioperative phase: (1) it lacks validation by an accepted and independent gold standard; (2) there is a wide spectrum of false-positive findings (considerable interindividual variations in left ventricular contraction, bundle branch blocks, hypertension, hypervolemia); (3) changes in the inferior and apical segments of the left ventricle cannot be detected by single-plane TOE. Detection in these segments might be achieved with biplane echocardiography, but few data on this improved technique are presently available; (4) the method is semi-invasive and might be not applicable during periods with a high incidence of myocardial ischaemia, e.g., intubation, the end of anaesthesia, and extubation; (5) anaesthetists seldom fulfil standard guidelines in echocardiography training; and (6) the method is expensive, which also limits its broader application. Cardiokymography, a noninvasive technique, allows analog representation of anterior wall motion.(ABSTRACT TRUNCATED AT 400 WORDS)


[Ventilatory changes during laparoscopic cholecystectomy]. During the past 2 years in Europe and the USA laparoscopic cholecystectomy (LC) has become a widely practiced procedure. Nevertheless, the effects of long-lasting laparoscopic procedures on carbon dioxide elimination have not yet been systematically investigated. METHODS. Approval from the institutional research review board was obtained, as was written informed consent from the patients. Eleven patients undergoing LC were studied. Patient age ranged from 31 to 67 years. All patients received total intravenous anaesthesia (fentanyl, propofol, vecuronium, DHB). Controlled ventilation with a tidal volume of 12-14 ml/kg was administered. Before introduction of anaesthesia a cannula was inserted into the left radial artery. Blood gas analysis was undertaken just before introduction, just before insufflation of carbon dioxide, and thereafter at two intervals, after reaching an intra-abdominal pressure of 12 mm Hg, 15 min apart. Oxygen consumption and carbon dioxide output were measured using a calorimeter (Deltatrac TM, Datex). Intra-abdominal pressure was maintained at 12 mm Hg during the operation. RESULTS. After onset of the pneumoperitoneum inspiratory peak and plateau pressure showed an increase by more than 40%. During the operation respiratory minute volume had to be increased by about 30-40% to maintain normocapnia. Oxygen consumption remained nearly unchanged during the procedure while carbon dioxide output increased up to 38% 60 min after onset of the pneumoperitoneum. D(a-A) CO2 showed no significant change, indicating no increase in dead space. Beginning with the insufflation there was a significant increase in mean arterial pressure that lasted until the end of the procedure. CONCLUSION. The described effects of carbon dioxide insufflation, especially the extent of carbon dioxide resorption, define the need for careful monitoring of respiratory function during LC, especially in patients with preexisting cardiopulmonary disease.


[Pharmacodynamic effects of S-(+)-ketamine on EEG, evoked potentials and respiration. A study in the awake dog]. Until recently, only the racemic mixture of ketamine has been used in anaesthesia. Little is known of the central nervous effects of the pharmacologically more potent S(+)-isomer. Information in regard to the putative receptor site involved in the mediation of its anaesthetic/analgesic effect is particularly sparse. METHODS. In order to evaluate the anaesthetic and antinociceptive properties of S(+)-ketamine, a dose-response relationship of the compound on the EEG, somatosensory-evoked potentials (SEP), and respiration was established. Increasing doses (2, 5, 10, 20 mg/kg) were given to trained and awake dogs (n = 10) at 10-min intervals. In order to detect a possible opioid receptor-related interaction, an antagonist of the methoxymorphinane series (cyprodime 80 g/kg i.v.) with higher selectivity than naloxone for the mu-receptor was given at the end. RESULTS. Compared to controls, S(+)-ketamine induced a dose-related increase in output in the theta-(3-8 Hz) band and an increase in output in the alpha-domain (8-13 Hz) following 20 mg/kg. Both effects were reversed completely by the opioid antagonist. At low doses (2-5 mg/kg) there was an increase in output (P less than 0.05) in the beta-(13-30 Hz) and a concomitant decrease in output (P less than 0.05) in the delta-(0.5-3 Hz) band. These effects were reversed with increasing doses (5-10 mg/kg). After 20 mg/kg, however, output in the delta-domain increased while power in the beta band decreased significantly (P less than 0.005) when compared to controls. Both effects were reversed by the opioid antagonist. Compared to controls, the reversal resulted in a 12% increase in output in the beta- and a 49% decrease in output in the delta-domain. In SEP, S(+)-ketamine induced a dose-related increase in peak latency and depression of amplitude of more than 50% when compared to controls. While latency changes were completely reversed, amplitude height was only partly restored by the antagonist. Respiration was depressed in a dose-related fashion (PaO2 decreased, PaCO2 increased). Hypoxaemia was fully reversed by the antagonist; hypercapnia was only partly reversed. CONCLUSION. The results support the presumption that the S(+)-isomer of ketamine induces opioid mu-receptor-mediated central effects. Hypersynchronisation of the EEG suggests a deep plane of anaesthesia after S(+)-ketamine. The pronounced blockade of impulses in the sensory nervous pathways suggests an efficient analgesic effect that is partly mediated by the opioid-receptor. The respiratory depression may be of importance when S(+)-ketamine is used in high dosages in man.


[Humidification and heating of anesthetic gases during pediatric anesthesia using the Cicero Anesthesia Workstation]. A series of 52 infants underwent general or urological surgery; all were ventilated with the CICERO. Two different flows of fresh gas were used. In group I (n = 21) the fresh gas flow was set exactly at the level of the minute volume, representing a half-open, non-rebreathing system. In group II (n = 31) the fresh gas flow was adjusted to 10% of the required minute volume. Temperature and relative humidity of the inspired gas were measured continuously close to the tracheal tube. Anaesthesia was accomplished with 2 vol% isoflurane, 21-30 vol% oxygen in nitrous oxide. The results were compared with those achieved with our time-tested paediatric equipment, a SERVO 900D ventilator with a Fisher-Paykel humidifier (Group III, n = 35). Using a high fresh gas flow, no increase in relative humidity in the inspired gas could be detected. The values varied between 12% and 25% (group I). Reducing the flow of fresh gas as indicated above resulted in an increase in the relative humidity (group II). Over the evaluated period of 2 h, humidity increased slowly from an initial mean value of 20% to a maximum of something over 70%. Using the SERVO 900D ventilator combined with the Fisher-Paykel humidifier, humidity reached a value of greater 90% within 10 min after activation of the heated cascade. Humidity in the inspired gas should exceed 70% to avoid damage to infant airways. This will not be attained until after more than 2 h with unaided breathing systems, by when most operations performed on paediatric patients will already be over. Condensed water may aspirated by small infants. This potentially dangerous situation was only encountered in the CICERO circuit, and not in the system protected by the Fisher-Paykel cascade. Dry gases can result in thickened mucous and in obstruction of a small tracheal tube, which requires emergency reintubation. With artificial airways dry gases damage the ciliated epithelia of the trachea and cause loss of water and body heat. The temperature of the "cold" gases varied within a range of 21-33 degrees C and could not be adjusted by the anaesthetist. In the CICERO system, heating the gases at the valve only prevents mechanical failure caused by water condensation. In pediatric anaesthesia, variable heating and non-condensing humidity are essential. The dry and heated gases of the CICERO are not acceptable in the daily practice of paediatric anaesthesia.


[Postspinal headache. A comparison of the 24G Sprotte syringe and a 29G Quincke needle]. A randomised study was performed to compare the frequency of postdural puncture headache in 56 patients who underwent spinal anaesthesia for extra-corporeal shockwave lithotripsy using either a Sprotte 24 G (n = 28) or Vygon 29 G or Quincke type needle (n = 28). Frequency of headache was recorded in a similar group of 28 patients who received general anaesthesia. Dural puncture was easier with the Sprotte 24 G cannula than with the less stable Quincke needle, as documented by a significantly shortened time for insertion of the cannula (4.6 +/- 2.6 vs 8.6 +/- 6.3 min, P less than 0.005). The total frequency of post-operative headache was 57% in the Vygon 29 G group and 25% in the Sprotte 24 G group; 21% of patients in the general anaesthesia group complained of headache. Frequency of postdural puncture headache, classified as being posture-related, was 25% in the 29 G Vygon group, compared with 11% in the 24 G Sprotte group (P = 0.148). When only moderate and severe postdural puncture headache was considered, there was a significant difference (25% vs. 4%; P = 0.026) in favour of the Sprotte cannula. Thus, the 24 G Sprotte needle was at least as effective as the 29 G Vygon needle, and there is a suggestion that the former is more effective in minimising the incidence of moderate or severe postdural puncture headache.


[Spinal endoscopy in the detection of problems caused by continuous spinal anesthesia]. Continuous spinal anaesthesia has a number of advantages, but there are a number of drawbacks as well: difficulties in threading the catheter, distribution of the local anaesthetics and the development of cauda equina syndrome. Spinaloscopy was done to visualize the fate of catheters during and after their insertion, as well as the distribution of local anaesthetics injected through these fine-bore catheters. METHOD. The studies were conducted in preserved and fresh cadavers donated to the Anatomic Institute for Medical Studies. The spinal column from T12 to S1, together with the back musculature (in order to preserve the normal curvature of the spine) were removed from the cadaver. Spinaloscopy was done with a 4 mm endoscope with a 0 degree optic (Storz, Tutlingen, FRG). All observations were made from the lumbosacral region of the dissected preparation. In this fashion, it was possible to observe the insertion of the spinal needle used to introduce the catheter into the subdural space. The distribution of local anaesthetics injected through a 22-gauge spinal needle or a 28-gauge catheter was shown by injecting 0.5% hyperbaric bupivacaine colored with a small amount of 1% methylene blue. Pictures were taken 15, 30 and 45 s after beginning the injection. RESULTS. Difficulty in threading the catheter: our observations indicate that the difficulty in inserting microbore catheters is most likely due to inserting the needle too far. It is impossible for the catheter to bend and be inserted into the subarachnoid space. In many cases the catheter encountered the anterior wall of the spinal canal and would slide along various structures. Distribution of the drug: the injection is better dispersed with a 22-gauge needle and it completely fills the subarachnoid space. The local anaesthetics injected through the 28-gauge nylon catheter (Kendall Healthcare, Mansfield, Mass.) are distributed in the dependent portions of the spinal canal. If high doses and a high concentration are injected, the distribution pattern may result in an overconcentration in some parts of the subarachnoid space. Possibility of trauma: the catheter stretches around the roots, the potential for trauma is that untoward stress may be applied to the root, either during full insertion of the catheter or during its withdrawal. CONCLUSION. Spinaloscopy was done in a non-fixated anatomic spinal column preparation with a 4 mm 0 degree endoscope (Storz, Tuttlingen, FRG). Based on our observations, we conclude: The catheter should only be inserted 2 cm into the subarachnoid space. This may decrease the risk of malpositioning. After the tip of the catheter has reached the subarachnoid space, the stylet should be with drawn 2 or 3 cm to minimize the risk of nerve injury and/or bleeding.


[The effect of adding clonidine to mepivacaine. Axillary brachial plexus blockade]. Clonidine (Cl) added to local anaesthetics (LA) prolongs the duration of both anaesthesia and analgesia after peripheral nerve blocks. In this study, we investigated the dose-dependent effect of Cl added to mepivacaine (M) on clinical efficacy, onset, and regression time of brachial plexus block. METHODS. Ninety patients were randomly assigned to one of three groups. Every patient received 46 ml of a mixture containing 400 mg M (pH adjusted with NaHCO3 to 7.25) and either 0.9% NaCl (group A), 0.12 mg Cl (group B), or 0.24 mg Cl (group C). The axillary block was performed using the catheter technique. In a double-blind fashion, the onset of sensory and motor blockade was tested every 5 min for 30 min. Duration of anaesthesia, analgesia, and motor blockade (time between injection and return of sensation, onset of pain, or ability to move, respectively) was assessed using a questionnaire. M plasma levels were measured by HPLC in 10 patients from each group for up to 120 min. Blood pressure (BP), heart rate (HR), and respiratory rate (RR) were measured for up to 120 min. Sedation was assessed using a verbal rating scale. RESULTS. There was no difference in the onset of blockade. There was dose-dependent prolongation of the duration of anaesthesia, analgesia, and motor blockade with significant differences between groups C and A regarding all three parameters, between groups C and B regarding duration of anaesthesia, and between groups B and A regarding duration of analgesia. There was no significant difference in M plasma levels. Although there was only a slight but significant decrease in mean BP values in groups B+C and no difference in HR and RR, 2 patients (1 group B, 1 group C) had marked decreases in BP and HR (less than 70 mmHg systolic resp. less than 50/min) after 120 and 210 min. Sedation occurred in most patients receiving Cl. CONCLUSIONS. Addition of Cl to LA produces a dose-dependent prolongation of anaesthesia, analgesia, and motor blockade. Neither the onset time nor the number of patients with adequate surgical anaesthesia was influenced by Cl. Considering the M plasma levels, it is unlikely that the prolongation of the block is caused by local vasoconstriction, which is proposed to be the mechanism of action of epinephrine. The mean differences in haemodynamic parameters were not of clinical relevance, but the two dramatic drops in BP and HR, probably caused by Cl, were significant.


[Surfactant administration in acute respiratory failure]. We report the case of a 21-year-old man who developed adult respiratory distress syndrome (ARDS) after severe lung contusion due to a car accident. At the scene of the accident the patient was awake and oriented, but there were signs of hypoxaemia (SaO2 by pulse oximetry: 86%). The trachea was intubated in the emergency room and, after diagnosis of multiple rib fractures on the right side (the patient nos. 4-11) and emergency treatment, was extubated 16 h later. During spontaneous breathing there was no improvement of pulmonary function, and the patient was transferred to the intensive care unit 5 days later and reintubated because of acute respiratory failure. He then developed the signs of severe ARDS. No improvement occurred during conventional ventilatory treatment including inversed-ratio ventilation, high-frequency ventilation, and ventilation via a double-lumen tube. On day 15 a bovine surfactant preparation (38 mg/kg body wt.) was instilled into both lungs. Initially there was deterioration of the pulmonary function, probably due to crusts in the bronchial mucous membrane. After aspiration of the crusts at bronchoscopy, there was progressive respiratory improvement. The inspired oxygen concentration and PEEP level could be reduced, and the ventilatory ratio normalised within 14 days. This therapeutic intervention improved pulmonary function and probably led to the successful outcome after 36 days of ventilatory support.


[Verification of the endotracheal tube placement]. Verification of the correct position of the endotracheal tube is a daily routine task of every anaesthesiologist. Accidental intubation of the oesophagus is a very rare complication in absolute terms but still the most frequent preventable anaesthetic mishap with fatal outcome. Even the most experienced anaesthetist is not immune to this complication. Only vigilance on the part of the anaesthetist protects the patient. There is no absolutely reliable gold standard to diagnose the correct position of the tracheal tube. Visualization of the endotracheal tube between the vocal cords and a typical CO2 excretion waveform are two of the best practical signs. After every change of position of the patient, especially after flexion or extension of the head, the position of the tube must be checked again. The old aphorism is still valid: When in doubt, take it out.


The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method. Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.


Lectin affinity electrophoresis for the separation of fluorescently labeled sugar derivatives. Lectin affinity electrophoresis was applied to the separation of charged, fluorescent conjugates of disaccharides. Four fluorescent conjugates were prepared by reductive amination of alpha-D-Man-(1----3)-D-Man, alpha-D-Gal-(1----4)-D-Glc, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc in the presence of 7-amino-1,3-naphthalenedisulfonic acid. These charged fluorescent-disaccharide conjugates all have identical molecular weight and in the absence of conconavalin A lectin failed to separate either by agarose or by polyacrylamide gel electrophoresis. In the presence of either free or immobilized concanavalin A, agarose gel electrophoresis and polyacrylamide gel electrophoresis could separate the fluorescent conjugate of alpha-D-Man-(1----3)-D-Man from that of alpha-D-Gal-(1----4)-D-Gal, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc.


Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates. In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.


Alkyl- and aryl-substituted salicyl phosphates as detection reagents in enzyme-amplified fluorescence DNA hybridization assays on solid support. Nine salicyl phosphate esters with hydrophobic substituents (5-phenyl, 5-(2,4-difluorophenyl), 5-tert-octyl, 5-cumyl, 5-(4-tert-butylphenyl, 5-(1-adamantyl), 5-(n-dodecyl), 5-(1,1-diphenylethyl, and 5-trityl) were synthesized and found to be good substrates for calf intestinal alkaline phosphatase. The enzymatic hydrolysis produced the corresponding salicylates, which were strongly fluorescent when excited by ultraviolet light around 300 nm with maximum emission at 420-435 nm. The salicylates were less soluble and/or more adhesive than the nonfluorescent salicyl phosphate substrates, resulting in localization of fluorescence signal, which is a requirement for membrane-based assays. The salicyl phosphates bearing 8-14 carbon substitutents were found to be suitable detection reagents for dot-blot DNA hybridization assays on nylon membrane using a biotinylated probe, allowing the detection of 125 pg of target pBR322 plasmid DNA using a simple apparatus consisting of a transilluminator, a camera. and a 455-nm cutoff optical filter.


The development of high-performance liquid chromatographic analysis of allyl and allyloxycarbonyl side-chain-protected phenylthiohydantoin amino acids. Ten phenylthiohydantoin (PTH) amino acids possessing allyl (Al) or allyloxycarbonyl (Aloc) side-chain-protecting groups have been characterized by high-performance liquid chromatography for use in Edman degradation sequence analysis. Optimized separation of side-chain-protected and -unprotected PTH amino acids was achieved on a C-18 reversed-phase column with a two-step gradient spanning 32 min. Five of the side-chain-protected amino acids [Cys(Al), Cys(Aloc), Lys(Aloc), Thr(Aloc), Tyr(Al)] were completely stable to the conditions of PTH derivatization, four [Asp(OAl), Arg(Aloc)2, Glu(OAl), Ser(Aloc)] were partially deprotected during PTH derivatization, and one [His(Aloc)] was completely deprotected during PTH derivatization. All allyl-based derivatives were well resolved from their side-chain-unprotected counterparts. Studies on the stability to piperidine treatment showed Asp(OAl), Cys(Al), Glu(OAl), Lys(Aloc), Thr(Aloc), and Tyr(Al), and possibly Arg(Aloc)2 and Ser(Aloc), to be suitable for peptide synthesis by 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry. Edman degradation of Al and Aloc side-chain-protected Conus geographus Lys9-alpha-conotoxin GI synthesized on 4-methylbenzhydrylamine-copoly(styrene-1%-DVB)-resin demonstrated the usefulness of these derivatives for solid-phase preview sequence analysis.


Two-dimensional differential scanning calorimetry: simultaneous resolution of intrinsic protein structural energetics and ligand binding interactions by global linkage analysis. A general theoretical development for the design and analysis of two-dimensional thermal stability surfaces of proteins is presented. The surfaces are generated from multiple excess heat capacity profiles (<delta Cp> vs T) obtained at varying concentrations of an interacting ligand. The energetics of both the intrinsic protein stability and the protein-ligand interaction are simultaneously resolved by employing statistical thermodynamic models in global linkage analysis. This formalism allows resolution of the intrinsic protein folding-unfolding parameters (enthalpy, entropy, and heat capacity changes) as well as the ligand interaction parameters (binding stoichiometry, enthalpy, entropy, and heat capacity changes). The theory has been applied to the case of ribonuclease A and its interaction with cytidine-2'-monophosphate. The accuracy of the thermodynamic parameters obtained by this approach compares within error with those parameters that can be obtained by direct measurements.


A solid-phase screen for protein kinase substrate selectivity. Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites. The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 6(7) approximately 2.8 x 10(5) sequences). The cassette was inserted into the gene of a suitable carrier protein and expressed in E. coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters. When the library was screened with TPK1 delta, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified. By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1 delta was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser). Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity.


A strategy for the mapping of N-glycans by high-pH anion-exchange chromatography with pulsed amperometric detection. We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of less than 0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients "S" and "A" for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of less than +/- 0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of less than +/- 1%, three of them even with an accuracy of less than 0.1%, providing the reliability of the established HPAE-PAD mapping database.


Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. A procedure is described for computing sedimentation coefficient distributions from the time derivative of the sedimentation velocity concentration profile. Use of the time derivative, (delta c/delta t)r, instead of the radial derivative, (delta c/delta r)t, is desirable because it is independent of time-invariant contributions to the optical baseline. Slowly varying baseline changes also are significantly reduced. An apparent sedimentation coefficient distribution (i.e., uncorrected for the effects of diffusion), g*(s), can be calculated from (delta c/delta t)r as [formula: see text] where s is the sedimentation coefficient, omega is the angular velocity of the rotor, c0 is the initial concentration, r is the radius, rm is the radius of the meniscus, and t is time. An iterative procedure is presented for computing g*(s)t by taking into account the contribution to (delta c/delta t)r from the plateau region to give (delta c/delta t)corr. Values of g*(s)t obtained this way are identical to those of g*(s) calculated from the radial derivative to within the roundoff error of the computations. Use of (delta c/delta t)r, instead of (delta c/delta r)t, results in a significant increase (greater than 10-fold) in the signal-to-noise ratio of data obtained from both the uv photoelectric scanner and Rayleigh optical systems of the analytical ultracentrifuge. The use of (delta c/delta t)r to compute apparent sedimentation coefficient distributions for purposes of boundary analysis is exemplified with an antigen-antibody system.


Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors. Inducible phosphorylation or dephosphorylation of transcription factors is an important mechanism of signal-dependent gene regulation in eukaryotic cells. This paper describes a combination of techniques that can be used to study the effect of this covalent protein modification on the DNA-binding activity of transcription factors. The protein of interest is genetically tagged with oligohistidine to allow rapid purification on nickel-chelate columns after being transiently overexpressed in eukaryotic tissue culture cells. Phosphorylation-dependent DNA-binding activity is determined in a blotting assay including an in situ dephosphorylation step. Studies on the protooncogene-encoded transcription factor c-Jun employing this assay revealed a TPA-inducible protein dephosphorylation event that strongly increases the DNA-binding potential of the protein. Our data confirm the results published recently by others and provide a rapid, efficient, sensitive, and well-controlled experimental system to analyze the phosphorylation-regulated modulations in the DNA-binding activity of transcription factors.


Development of a radiometric spot-wash assay for squalene synthase. The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1-14C]isopentenyl diphosphate as a precursor for squalene or [2-14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2-14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughout mode or in a qualitative, high-through-put mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.


Preparation of iron-deficient tissue culture medium by deferoxamine-sepharose treatment and application to the differential actions of apotransferrin and diferric transferrin. We have shown that triiodothyronine-dependent GH1 rat pituitary cell growth in serum-free defined culture required apotransferrin (apoTf) (D. A. Sirbasku, et al., Biochemistry 30, 295-304, 7466-7477, 1991). These studies were done in "low-Fe" medium without Fe(III)/Fe(II) salts. Nonetheless, significant concentrations of iron may have been contributed by other components, making this medium unsuitable for study of the differential effects of apoTf and diferric transferrin (2Fe.Tf). Measuring residual iron in culture medium has been troublesome because the most sensitive method (i.e., atomic absorption) detected levels only in excess of 10 ng/ml and did not distinguish between the forms of iron present. To estimate the Fe(III) available to bind to apoTf, we developed a more sensitive and specific method. Urea-polyacrylamide gel electrophoresis (PAGE) separates apoTf, the two monoferric transferrins, and 2Fe.Tf. [125I]apoTf was incubated with medium, or components, and the formation of [125I]-2Fe.Tf was monitored by urea-PAGE/autoradiography. By this method, the concentration of Fe(III) in low-Fe medium was estimated at 8.4 to 20 ng/ml and the sources were identified. We next sought to remove the Fe(III). Standard chelators were ineffective or cytotoxic. In contrast, an affinity method with deferoxamine-Sepharose depleted greater than or equal to 90% of the Fe(III). In this medium, apoTf and 2Fe.Tf showed differential effects with GH1 cells and with MCF-7, MTW9/PL2, an MDCK cells. With the methods described here, the effects of apoTf and 2Fe.Tf on growth can be studied separately.


Immunochemical detection of proteins and nucleic acids on filters using small luminescent inorganic crystals as markers. A new luminescent marker for the immunochemical detection of proteins and nucleic acids on filters is reported. The label consists of inorganic crystals, generally called phosphors, with a particle size of 0.1-0.3 microns, stabilized in suspension with polycarboxylic acids and subsequently conjugated to immunoreactive macromolecules. Immunophosphor conjugates exhibit slowly decaying fluorescence that is strong and practically nonfading and not sensitive to quenching by water molecules. They are therefore suited for conventional fluorescence detection as well as for time-resolved detection. The lifetime of the phosphors was in the micro/milliseconds range upon excitation with ultraviolet light. Proteins or nucleic acids immobilized on nitrocellulose filters were detected immunochemically or by hybridization, using haptenized nucleic acid probes followed by immunochemical detection, respectively. The ultimate detection limit of proteins, using phosphor-labeled macromolecules including an immunochemical amplification step, was found to be 10 fg. The detection limit of nucleic acids was 300 fg for demonstration of hapten-labeled probes and 10 pg in hybridization formats with hapten-labeled probes. The sensitivity of methods using phosphor-labeled macromolecules was in all cases as good as or better than that of methods using alkaline phosphatase developed to NBT/BCIP. The use of immunophosphors for detection of proteins and nucleic acids on Western and Southern blots is demonstrated. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.


An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides. A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.


In vitro transcription by RNA polymerase II in extracts of Xenopus oocytes, eggs, and somatic cells. We describe procedures for preparing extracts of Xenopus oocytes, eggs, and somatic cells that will accurately transcribe class II genes. A variety of viral and Xenopus promoters direct the accurate initiation of transcription by RNA polymerase II in these extracts. Optimal ionic conditions (100-200 mM KCl, 12 mM MgCl2), template concentration (20-40 micrograms/ml), incubation time (30-60 min), and temperature (25 degrees C) for class II gene transcription are described.


Gas chromatography/mass spectrometry assays for the determination of debrisoquine and sparteine metabolites in microsomal fractions of rat liver. Debrisoquine and sparteine are prototype substrates of a genetic deficiency in cytochrome P450-dependent drug metabolism. Sensitive assays of in vitro oxidation of sparteine and debrisoquine are required for evaluation of this polymorphism. The activities were measured by quantitative analysis of 2-dehydrosparteine and 4-hydroxydebrisoquine production, respectively, using capillary column gas chromatography coupled with mass selective ion detection. With a single extraction, separation of parent drug, metabolite, and a suitable internal standard was readily achievable. Time-dependent production of both metabolites could be detected from as little as 40 micrograms of microsomal protein. Both activities showed a maximal activity with a 240-min incubation period. The ability to simultaneously quantify the parent drug and its metabolite suggests it would also be useful for evaluation of in vivo metabolism.


Ring test for low levels of N-(2-hydroxyethyl)valine in human hemoglobin. Hemoglobin adducts are useful for the identification and quantification of electrophilic agents in vivo. A modified Edman degradation method has been extensively used for monitoring exposure to ethylene oxide through gas chromatographic-mass spectrometric measurements of hydroxyethyl adducts to the N-terminal valines in hemoglobin. In a ring test, four laboratories using different versions of the method analyzed eight human globin samples with low adduct levels from ethylene oxide. Measurements of the same adduct by a radioimmunoassay were also included. Strong correlation between the measurements by the different laboratories shows that the method in principle works well. However, there were some systematic quantitative differences.


Bioluminescence detection system of mutagen using firefly luciferase genes introduced in Escherichia coli lysogenic strain. A rapid and convenient microbial sensing system for mutagens was developed based upon the induction of prophage from Escherichia coli lysogenic strain and bioluminescence. The system consisted of lysogenic E. coli encoding firefly luciferase genes and a photodetection system. Measurement of mutagen mitomycin C was achieved by measuring the luminescence intensity emitted from E. coli lysogenic strain for the recombinant phage in the presence of luminescence substrates. Approximately 1 h after addition of mitomycin C, the luminescence began to be observed, and 3 h after, it attained a level of 2 times greater than that of 1 h. Irradiation with ultraviolet light also produced light based on induction of phage from the E. coli lysogenic strain for the recombinant phage. On the other hand, when nonmutagenic toxic compounds like sodium azide were added to the reaction medium, luminescence was not observed. Mitomycin C could be detected within 1 h with this sensing system, at concentrations down to 10(2) ng/assay.


Reversible potentiometric oxygen sensors based on polymeric and metallic film electrodes. Various materials and sensor configurations that exhibit reversible potentiometric responses to the partial pressure of oxygen at room temperature in neutral pH solution are examined. In one arrangement, platinum electrodes are coated with plasticized poly(vinyl chloride) films doped with a cobalt(II) tetraethylene pentamine complex. For such sensors, potentiometric oxygen response is attributed to a mixed potential originating from the underlying platinum electrode surface as well as a change in redox potential of the Co(II)-tetren-doped film as the complex binds oxygen reversibly. The response due to the platinum surface is prolonged by the presence of the Co(II)-tetren/PVC film. Alternately, thin films of metallic copper, electrochemically deposited on platinum and/or sputtered or vapor deposited on a single crystal silicon substrate, may be used for reversible oxygen sensing. The long-term reversibility and potentiometric stability of such copper film-based sensors is enhanced (up to 1 month) by preventing the formation of cuprous oxide on the surfaces via the application of an external nonpolarizing cathodic current through the working electrode or by specifically using sputtered copper films that have [100] preferred crystal structures as determined by X-ray diffraction. The implications of these findings in relation to fabricating analytically useful potentiometric oxygen sensors are discussed.


Enzyme packed bed system for the on-line measurement of glucose, glutamate, and lactate in brain microdialysate. Rapid measurement of glucose, glutamate, and lactate is important in understanding the dynamics of the energy balance of the brain. Glutamate is also the main excitatory neurotransmitter. A general immobilized enzyme-based flow injection assay system is described which uses oxidase and peroxidase enzymes to convert the analyte into an oxidized ferrocene species which is detected electrochemically by reduction. The enzymes glucose oxidase, glutamate oxidase, lactate oxidase, and horseradish peroxidase are immobilized with near 100% efficiency onto 10-microns tresyl-activated silica beads (1000- and 500-A pore size). The beads are slurry-packed into 2- x 20-mm columns to give beds for glucose, glutamate, or lactate which are stable for greater than 40 days. The flow injection assays described have detection limits from 1.8 to less than 20 pmol and have been configured to have linear calibration responses over the range of basal and stimulated levels of the three compounds found in 5-microL microdialysate samples from the rat striatum. The assays are used for automated on-line measurement of glucose, glutamate, and lactate in striatal microdialysate at 2.5-min intervals.


Comparison of BaTiO3 optical novelty filter and photothermal lensing configurations in photothermal experiments. Photorefractive BaTIO3 is used as an optical novelty filter to highlight the high spatial frequency components of the photothermal signal. A real-time phase grating recorded in BaTIO3 acts as a matched rejection spatial filter for the probe laser. This reduces the stationary background from the optical signals thereby increasing signal contrast ratios. Rejection of the monotonous stationary signal provides a powerful means of improving the photothermal signal. This paper describes the construction of this novel apparatus and the experiments performed in order to compare its performance with photothermal lensing results. A theory that explains photothermal signal filtering with BaTIO3 as an adaptive spatial frequency filter is presented. Results comparing the optical signals obtained in a photothermal lensing experiments and those obtained in the BaTIO3 optical novelty filter experiments are presented. The optical novelty filter signals demonstrate a remarkable improvement in the signal contrasts for moderate photothermal-induced phase shifts.


Effect of aging the hollow cathode by sputtering on the analytical precision of the hollow cathode discharge emission source. Sputtering changes the analytical performance of the hollow cathode as an emission source. Proper conditioning (aging) of the hollow with sputtering greatly improves precision. Conditioning decreases the blank emission signal and changes the shape of the hollow. Micrographs which show the evolution of the bottom of the hollow into a stable bulb shape are presented. The blank emission signal decreases with aging of the hollow and approaches a constant value when the hollow is properly conditioned. A short-term stability (n = 5) of approximately 1.8% RSD has been observed frequently. An average long-term stability over several days (n = 45) of 4.5% for Li and 3.5% for Na in the emission signal from microsamples (less than 50 nL) deposited in the hollow cathode discharge source is reported here. Instrumentation, operation, and sample preparation procedures are described.


Capillary electrophoresis-electrospray mass spectrometry for the analysis of recombinant bovine and porcine somatotropins. A Beckman P/ACE 2050 high-performance capillary electrophoresis (HPCE) instrument has been interfaced with a Vestec electrospray ionization (ESI) mass spectrometer for the analysis of recombinant proteins. Peak resolution is not compromised by coupling HPCE to an ESI mass spectrometer. Recombinant bovine and porcine somatotropins (rbSt and rpSt) were used as model proteins. The standard curve of the capillary zone electrophoresis (CZE) method with UV detection for the determination of rpSt is linear in the range of 7-300 fmol with theoretical plates of approximately 410,000 m-1. The relative standard deviation for the rpSt peak migration time is less than 1%. The multiply-charged ion clusters obtained in the CZE-ESI mass spectrum for a sample of rpSt ranged from mlz 1363.2 (the cluster with 16 charges) to 1982.5 (the cluster with 11 charges). The average molecular weights of 21,812.6 and 21,798.3 for a sample of rbSt and rpSt determined in this study were nearly identical to the theoretical values of 21,812.0 and 21,797.9, respectively. Detection limit of the CZE-ESI mass spectrometer is approximately 100 fmol. The CZE method separated mono- and dideamidated species and monoacetylated compounds while the ESI mass spectrometer detected an analogue and a truncated homologue of rpSt comigrating with the major peak. The presence of mono- and dioxidized homologues was also detected in the major peak of some rbSt and rpSt samples. These data clearly indicated that, individually, both CZE and ESI mass spectrometric methods could not detect all impurities. Coupling of the HPCE Instrument and the ESI mass spectrometer enhances analytical capabilities of both tools for rapid characterization of recombinant proteins.


Organic ion imaging using tandem mass spectrometry. A triple-quadrupole mass spectrometer has been interfaced with a wide-angle secondary ion microprobe. The combination permits acquisition of data necessary to determine the distribution of targeted organic analytes even in the presence of overwhelming isobaric interference. Micrographs generated from secondary ion intensity alone are compared to those generated using secondary ionization with tandem mass spectrometry (MS/MS), both for image reference and to show the improvement in image quality that can be attained when MS/MS is employed. Inhomogeneous mixtures of glycerol, KCl, and asparagine on 1-cm-diameter aluminum targets were used to demonstrate the instrument's selectivity. Secondary ions generated from samples of this system include isobaric 133Cs+ implanted from the primary ion beam, the 41K(+)-glycerol adduct, and protonated asparagine.


Controlling solvent strength and selectivity in micellar liquid chromatography: role of organic modifiers and micelles. Simultaneous enhancement in elution strength and selectivity which has been previously observed in micellar liquid chromatography (MLC) for a variety of compounds is further investigated. The reasons behind the occurrence of this unique phenomenon are studied, and the influence of micelles and organic solvents on elution strength and selectivity is discussed. A model is developed which explains the dependence of the solvation ability of organic solvents in MLC (represented by the solvent strength parameter, S, of solutes) and the degree of solute interactions with micelles. Whenever the difference in solvent strength parameter values of two solutes in micellar eluents, dS, is positive, maximum selectivity is observed at the weakest eluent strength. When dS less than 0, there exists an inverse relationship between retention and solvent strength parameter so that selectivity monotonically increases with volume fraction of organic solvent in micellar eluents. It is shown that usually there is no direct relationship between the solvent strength parameter in MLC and retention. As a result, selectivity enhancement due to an increase in the concentration of organic modifier (i.e. solvent strength) occurs frequently in MLC. Interestingly, for cases where selectivity decreases with an increase in organic modifier, simultaneous enhancement of selectivity and solvent strength can be observed by increasing micelle concentration. In a sense, the concentrations of organic modifier and micelles complement one another in improving selectivity at higher elution strengths. As a result of this unique phenomenon better separations in shorter analysis times can be observed. The mutual effects of micelles and organic modifier on one another would also require a simultaneous optimization of these two parameters.