Time and cost analysis of a computer-assisted telephone interview system to collect dietary recalls.

This study, conducted in 1991, examined the time requirements and costs of obtaining 24-hour dietary recalls via telephone interviews using the University of Minnesota's microcomputer Nutrient Data System to conduct the interviews and compute the nutrient composition of the diets. The subjects were 156 hypercholesterolemic children (aged 4-10 years) and 102 hypercholesterolemic adults (aged 21-65 years), who were participating in ongoing cholesterol education programs. A total of 391 recalls were completed with the children and 278 with the adults. For each completed interview, 3.5 and 2.8 attempts were required, respectively. Evenings were the most productive time for completing interviews. All tasks associated with completing the interviews (attempts to call, interviews per se, and postinterview procedures) required an average of 39.7 and 35.5 minutes per completed interview with the children and adults, respectively. About half of these total times were actually devoted to conducting the interview. The costs per completed interview were $9.22 for the children versus $6.99 for the adults. This difference reflects the greater number of attempts required to reach the children, the longer duration of their interviews, and the higher intrastate toll rates for calls to them as compared with the interstate rates for calls to the adults.


Random digit dialing: the potential effect on sample characteristics of the conversion of nonresidential telephone numbers.

This study examined the changes over time in the residential status of telephone numbers and the characteristics of households with telephone numbers that changed from nonresidential to residential. The authors determined the status in 1987 of all phone numbers (n = 9,107) that had been found to be nonresidential in six case-control studies conducted between 1979 and 1986 in Washington State. A telephone interview to obtain information on household characteristics was completed for 1,333 of the 1,901 phone numbers that had become residential by 1987. The interviews revealed that households with phone numbers that had previously been nonresidential differed from the general population with respect to household income, the age of household members, and the education of the head of the household. Data for the same area showed that the proportion of all phone numbers that were residential changed differentially according to the prefix (which roughly defines geographic area) during a 12-month period between 1987 and 1988, with some prefixes showing increases in residential phone numbers of more than 20%, while other prefixes decreased or stayed the same over the same period. We conclude that methods of random digit dialing that use two-stage designs or exclude previously dialed numbers may result in biased sampling unless changes over time are considered.


Correction of logistic regression relative risk estimates and confidence intervals for random within-person measurement error.

Frequently, covariates used in a logistic regression are measured with error. The authors previously described the correction of logistic regression relative risk estimates for measurement error in one or more covariates when a "gold standard" is available for exposure assessment. For some exposures (e.g., serum cholesterol), no gold standard exists, and one must assess measurement error via a reproducibility substudy. In this paper, the authors present measurement error methods for logistic regression when there is error (possibly correlated) in one or more covariates and one has data from both a main study and a reproducibility substudy. Confidence intervals from this procedure reflect error in parameter estimates from both studies. These methods are applied to the Framingham Heart Study, where the 10-year incidence of coronary heart disease is related to several coronary risk factors among 1,731 men disease-free at examination 4. Reproducibility data are obtained from the subgroup of 1,346 men seen at examinations 2 and 3. Estimated odds ratios comparing extreme quintiles for risk factors with substantial error were increased after correction for measurement error (serum cholesterol, 2.2 vs. 2.9; serum glucose, 1.3 vs. 1.5; systolic blood pressure, 2.8 vs. 3.8), but were generally decreased or unchanged for risk factors with little or no error (body mass index, 1.6 vs. 1.6; age 65-69 years vs. 35-44 years, 4.3 vs. 3.8; smoking, 1.7 vs. 1.7).


Cystic kidney dysplasia and polydactyly in 3 sibs with Bardet-Biedl syndrome.

Two infants with cystic kidney dysplasia and polydactyly were born to consanguineous parents. One infant died at age 2 months, and the other is currently 3.5 years old. A third pregnancy was terminated following ultrasonographic visualization of large echo-dense fetal kidneys and polydactyly. Although none had apparent brain anomalies, they were considered to represent the Meckel syndrome. Extinguished responses on electroretinography in our 3.5-year-old patient has led to the diagnosis of Bardet-Biedl syndrome. This observation offers an opportunity to revisit the Bardet-Biedl syndrome and provides further evidence that structural renal abnormalities are characteristic of the syndrome. We wish to alert the clinician to the diagnosis of Bardet-Biedl syndrome in patients with infantile cystic kidney dysplasia.


Postaxial acrofacial dysostosis: report on two patients.

We report on 2 patients with the postaxial acrofacial dysostosis (AFD) syndrome. One patient was an isolated case; the other had an equally affected brother previously described [Richieri-Costa and Guion-Almeida, 1989]. Recurrence in sibs suggests autosomal recessive inheritance.


Amniotic band sequence: Streeter's hypothesis reexamined.

Recently published reports of 54 subjects with the amniotic band syndrome (ABS) were reviewed, paying particular attention to internal anomalies. Evidence from the internal anomalies suggests that in most cases reviewed, damage occurred in a definable time period, probably prior to 26 days postconception and before the establishment of effective embryonic circulation. Most defects are explicable in terms of interference with neuropore closure, malmigration of cephalic neural crest tissue, and damage to the mesonephros consistent with local interference of the graded expression of organizational genes resulting in a local defect in the organization of the embryo.


Portohepatic shunt in a Down syndrome patient with an interchange trisomy 47,XY,-2,+der(2),+der(21)t(2;21)(p13;q22.1)mat.

A rare vascular portohepatic anomaly was identified in a Down syndrome patient with a 47,XY,-2,+der(2),+der(21)t(2;21)(p13;q22.1) mat chromosomal complement. This vascular defect involves a direct communication between the right portal vein and the inferior vena cava (IVC). We discuss the possibility that this vascular defect is a rare manifestation in Down syndrome. Alternatively, the existence of these 2 rare events in the same patient raises the possibility that they are causally related.


Rambam-Hasharon syndrome of psychomotor retardation, short stature, defective neutrophil motility, and Bombay phenotype.

We describe 2 Arab patients, both offspring of unrelated consanguineous matings, with unusual facial appearance, severe mental retardation, microcephaly, cortical atrophy, seizures, hypotonia, dwarfism, and recurrent infections with neutrophilia. Neutrophil motility was markedly decreased but the opsonophagocytic activity was normal. Both patients lack the red blood cell (RBC) H antigen and manifest the Bombay (hh) phenotype. Familial endocardial fibroelastosis and familial tetralogy of Fallot segregated independently in one family. The occurrence of the same syndrome in 2 unrelated families suggests that the various aspects of the disorder are the pleiotropic effects of a single mutation. Homozygosity-by-descent for a deletion involving contiguous genes may explain the findings in this syndrome. Alternatively, a mutation which involves an ubiquitous GDP fucose donor rather than the enzyme (alpha 2-L-fucosyltransferase) or its substrate (glcNAc) may account for the pleiotropic manifestations in this syndrome.


Sling left pulmonary artery, bridging bronchus, and associated anomalies.

Sling left pulmonary artery (SLPA) is often associated with tracheobronchial abnormalities, including bridging bronchus (BB). We report on 3 patients with SLPA: One patient had a narrow trachea, absent right upper lobe and right main bronchus, and BB. The second patient had a long and narrow trachea, with normal segmentation of bronchial tree, abnormal cerebral gyri, and minor facial abnormalities. The third patient, with a normal trachea and main bronchi with BB, had imperforate anus, hemivertebrae, and atrial septal defect (VATER association). Patients with SLPA, those with BB, or those with both SLPA and BB as well as multiple congenital anomalies represent a spectrum of anomalies.


Asymmetry quantification utilizing hand radiographs.

Asymmetry can be either directional or fluctuating. Detection of abnormal amounts of asymmetry has important implications for clinical diagnosis, but measurement of subtle levels is very difficult. We describe a method and normative values for asymmetry quantification using hand radiographs.


Application of the Health Belief Model in a study on parents' intentions to utilize prenatal diagnosis of cleft lip and/or palate.

Parents of children with cleft lip and/or palate (42 women and 35 men) participated in a study on intentions to use prenatal diagnosis of cleft by ultrasound in subsequent pregnancies. Based on the Health Belief Model (HBM) [Rosenstock, 1974], parents' cognitions on 4 factors were measured by questionnaires: "susceptibility" and "severity perceptions," "benefits" and "barriers" evaluations. Most parents perceived the defect as severe. Over-estimation of recurrence risks was predominant even among parents who had received genetic counseling. Results showed that most parents intend to utilize prenatal diagnosis but do not intend to abort an affected fetus. Subjects' reported reasons represented 3 thematic categories: cognitive (the need to know), emotional, and behavioral. Parents' intentions to diagnose and to terminate were related to the factors predicted by the HBM model. Regression analyses indicated that 38% of the variance in intentions to diagnose and 56% of the variance in intentions to terminate could be explained by the studied variables. The best predictor of both intentions was the perceived benefits of the diagnosis. Implications of these findings for genetic counseling are discussed.


Acrometageria: a spectrum of "premature aging" syndromes.

A child with manifestations of acrogeria and metageria, two "premature aging" syndromes, is presented. Because of his indistinct phenotype and because the question has been previously raised as to whether these conditions are separate, we propose the designation of acrometageria to describe this phenotypic continuum. As there is much in common clinically between acrometageria and the syndrome of type III procollagen deficiency (Ehlers-Danlos type IV), it might be presumed that a similar pathogenesis for acrometageria exists. This possibility has been tested previously, without demonstrating specific quantitative or qualitative deficits, but with some indirect evidence that collagen metabolism is deranged in these patients. One such crude indicator is the elevation of urinary hyaluronic acid levels, demonstrated in our patient and also observed in the phenotypically distinct Werner and Hutchinson-Gilford premature aging syndromes. On one hand, it could be argued that this supports the concept that premature aging syndromes exist as a biological continuum. On the other hand, it is equally valid to argue that syndromes of premature aging are so described merely because they include recognizable changes of normal aging and that the demonstration of an underlying mutation in a collagen gene, for example, invalidates their study as models of accelerated normal aging.


Fluorescence in situ hybridization (FISH) of a whole-arm translocation involving chromosomes 18 and 20 with alpha-satellite DNA probes: detection of a centromeric DNA break?

Fluorescence in situ hybridization (FISH) with alpha-satellite DNA probes was used to study whole-arm chromosome translocation products in a family in which the propositus was shown to have a monosomy 18p/trisomy 20p imbalance. By this approach, we show that the chromosome 18 alpha-satellite DNA block is split into 2 smaller units, whereas the chromosome 20 breakpoint is not included within the alpha-satellite DNA region. We found no evidence to suggest that this split alpha-satellite DNA region has reduced or impaired the function of the centromere or that it contributed to the phenotype of the propositus. The FISH technique critically demonstrated the involvement of a whole-arm translocation in this case and provided accurate identification of breakpoints, which was not possible with standard banding techniques.


Innovative approach to genetic counseling services for the deaf population.

Genetic service providers have stressed the importance of genetic counseling that is nondirective and specific to the personal needs of consultants. Successful genetic counseling for deaf persons often requires special provisions for complex family histories, syndromic conditions, and diversity in communication methods and cultural orientation. The Gallaudet University Genetic Services Center (GSC) was established in 1984 to provide genetic education and counseling services to the deaf community. The GSC staff developed and implemented a standardized system of data collection (family and medical history), clinical evaluation by consultant clinical geneticists, and counseling in sign language. In addition to clinical services, an in-depth educational program for professionals and consumers was developed and carried out. During a 6-year period, over 220 educational presentations were made and 659 deaf persons were seen for genetic evaluation and counseling. Most of these persons were self-referred. Sign language was the preferred means of communication of more than 90% of these individuals. A genetic cause of deafness was diagnosed in over 50% of the deaf consultants and was confirmed by segregation analysis, which had results similar to those reported for other studies of students in schools for the deaf. Special materials and strategies were developed in order to provide genetic services that were sensitive to the cultural and linguistic differences of the deaf population. These included written and visual materials that contained culturally neutral terminology and training of all staff members in sign language and the culture of the deaf.


Tracheoesophageal fistula, gastrointestinal abnormalities, hypospadias, and prenatal growth deficiency.

We studied 2 sibs, born to consanguineous parents, who presented with an MCA pattern which includes low birthweight, tracheoesophageal fistula, duodenal atresia, extrahepatic biliary atresia, hypoplastic pancreas, and hypospadias. This constellation of congenital anomalies appears to be a previously unreported autosomal recessive syndrome. A computerized search of the data files of the Spanish Collaborative Study of Congenital Malformations (ECEMC) identified 3 other unrelated infants with intestinal atresias, hypospadias, and low birth weight. These cases may represent a milder expression of the same syndrome.


Clinical phenotype and molecular analysis of a three-generation family with an interstitial deletion of the short arm of chromosome 5.

We report on a 3-generation family with an interstitial deletion of the short arm of chromosome 5. Varied manifestations were found among the affected individuals including microcephaly, hypertonia, and micrognathia; mental retardation was common to all affected individuals. High resolution chromosome analysis was interpreted as del(5) (pter- > p14.3::p13.3- > qter). Molecular comparison of the deletion in this family with individuals with other 5p deletions suggests that the clinical findings are due specifically to the chromosomal material deleted from 5p13.


Triply discordant triplets: probability, management options, and risks.

The spontaneous occurrence of triplets is rare. With increased utilization of "assisted reproductive technologies," multifetal gestations have become more common. The empiric fetal risk for major malformation is approximately 3%. In a triplet pregnancy each fetus independently carries this risk so that the probability of having at least one malformed fetus is approximately 9%. It is much less likely to have 2 or 3 simultaneously but discordantly malformed fetuses in a multizygotic triplet gestation (.09% and .0027% risk, respectively). We report on the first case, to our knowledge, of an ovulation-stimulated triplet pregnancy complicated by 3-way discordance for major malformations diagnosed in the late second trimester by ultrasound. Fetus A was affected by congenital diaphragmatic hernia and trisomy 21; fetus B had encephalocele, a midline facial defect, and a cleft palate; and fetus C had evidence of unilateral claw hand but an otherwise normal fetal survey. At 19 weeks of gestation, fetus A was found to have spontaneously died, and a selective termination of triplet B was performed. We conclude: (1) the finding of a single major malformation in one fetus should lead to extensive search for malformations in all members of the pregnancy, and (2) the simultaneous occurrence of major malformations in more than one member of a multifetal gestation is a circumstance under which multiple selective termination deserves consideration. In this article we discuss important issues and caveats in the performance of selective termination for abnormal members of multifetal gestations.


Prognosis of prenatally diagnosed children with sex chromosome aneuploidy.

Sex chromosome aneuploidy (SCA) occurs in about 1/250 amniocenteses, and the significance of the long-term prognosis of fetuses with SCA is of concern to prospective parents and health care providers. Longitudinal studies in an unselected group of newborn infants with SCA diagnosed postnatally have refuted allegations of mental retardation but have documented an increased risk for developmental problems. Of the 530 phone consultations with parents faced with a prenatal diagnosis of SCA, 68% continued the pregnancy. Twenty of the oldest subsequently born children (now 7-14 years old) were available for follow-up. In this small sample and age group, the propositi are progressing developmentally at a rate comparable to their sibs and are doing better at school and in peer relations than the SCA group diagnosed postnatally. Only 2 have documented IQs as low as 90. The documented IQs of the remainder, none of whom are sex chromosome mosaics, are all over 110. The parent population in this prenatally diagnosed group is unique and different from that of the postnatally diagnosed group in that over 85% of them are college graduates, often professionals, and upper socioeconomic individuals. The developmental competence of this SCA sample may be attributable to the supportive environment provided by these families, all of whom made a conscious decision to continue the pregnancy.


Case of ovarian dysgenesis and dilated cardiomyopathy supports existence of Malouf syndrome.

We describe an 18-year-old girl with ovarian dysgenesis, dilated cardiomyopathy, mild mental retardation, broad nasal base, blepharoptosis, and minor skeletal abnormalities. This unusual association of manifestations was first reported by Malouf et al. [1985]. Our patient, although a sporadic case, supports the existence of Malouf syndrome.


Ectodermal dysplasia, lipoatrophy, diabetes mellitus, and amastia: a second case of the AREDYLD syndrome.

A 19-year-old female with ectodermal dysplasia, lipoatrophy, diabetes mellitus, and amastia is described. This complex of symptoms is very similar to that of a case published by Pinheiro et al [1983] under the acronym of AREDYLD syndrome.


Additional dystrophin fragment in Becker muscular dystrophy may result from proteolytic cleavage at deletion junctions.

Becker muscular dystrophy is usually caused by intragenic dystrophin gene deletions that result in production of an internally deleted protein. Previous studies have detected what appears to be a unique dystrophin degradation product that appears only in muscle biopsies from patients with Becker muscular dystrophy. This dystrophin fragment is always seen in addition to the "full-size" dystrophin of the expected size for a given gene deletion. It is only found in biopsies from patients with mutations in the deletion-prone region encompassing exons 45-53, but it does not appear to correlate with any observable phenotype at the clinical level. By correlating the size and locations of dystrophin gene deletions with the size of this degradation product, together with use of region-specific dystrophin antisera, we find that proteolytic cleavage may occur at the deletion breakpoints, perhaps due to alterations of the secondary and/or tertiary structures of the protein. This cleavage results in loss of the carboxy-terminal domains that are thought to be important for interactions between dystrophin and other membrane-bound proteins.


Glomerular disease and human immunodeficiency virus infection in Brazil.

Clinically overt glomerular disease was detected in 6 (1.1%) of 543 patients with HIV infection followed at a Brazilian National Referral Center for AIDS. In 4 cases, glomerulosclerosis was present (focal and segmental in 3, diffuse and global in 1) and rapid progression to terminal renal failure was observed 1-10 months after clinical presentation. The other 2 patients died with normal renal function, and autopsy studies suggested the diagnosis of minimal change disease. Clinically overt glomerular disease was significantly more common among Black patients, whether all the cases with glomerulopathy (p < 0.001) or just the cases with glomerular sclerosis were considered (p = 0.011). Autopsy study of renal fragments from patients without clinical evidence of glomerular disease was additionally performed and revealed the presence of focal and segmental glomerulosclerosis in 3 cases (7.5%). We concluded that a glomerulopathy with clinicopathological features which match the definition of HIV nephropathy can be found among Brazilian patients with HIV infection. Accordingly to what has been described in American series, Brazilian Black patients seem to be at increased risk of the development of that nephropathy.


Comparison of subcutaneous and intravenous recombinant human erythropoietin for anemia in hemodialysis patients with significant comorbid disease.

While recombinant human erythropoietin (rHuEPO) is an effective therapy for anemia in renal failure, most published studies concern benefits in relatively healthy hemodialysis patients. The present study compares intravenous and subcutaneous administration of rHuEPO in an unselected group of 128 hemodialysis patients who were randomized to receive rHuEPO in an initial dose of 150 U/kg/week in three divided doses by subcutaneous or intravenous injection. Following a 4-week placebo run-in period, patients received rHuEPO until their hemoglobin was stable between 105 and 125 g/l for 4 weeks and then followed for a further 24 weeks. Eighty-three patients completed the study, 45 in the subcutaneous and 38 in the intravenous group. There was no difference in mean hemoglobin at any stage between subcutaneous and intravenous patients. Mean rHuEPO dose at the time of stabilization was significantly lower in the subcutaneous group compared to the intravenous (205.9 +/- 135.4 vs. 274.1 +/- 142.4 U/kg/week; p = 0.019), mean time to hemoglobin target was 9.9 +/- 4.5 weeks for the subcutaneous group and 11.9 +/- 4.9 weeks for the intravenous group (p = 0.037). Time to stabilization was 14.9 +/- 4.7 weeks for the subcutaneous compared to 17.3 +/- 3.9 weeks for the intravenous group (p = 0.006). Diabetic patients had higher dose requirements for rHuEPO at all time points and required a longer time to reach stabilization than nondiabetics (18.6 +/- 4.6 vs. 15.6 +/- 4.3 weeks; p = 0.016). Quality of life estimated by a disease-specific Kidney Disease Questionnaire improved significantly during rHuEPO therapy in both groups. There was no significant change in dialysis prescription throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)


Long-term suppression of secondary hyperparathyroidism by intravenous 1 alpha-hydroxyvitamin D3 in patients on chronic hemodialysis.

The effect of intravenous 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3] on circulating levels of intact parathyroid hormone (PTH 1-84) and COOH-terminal immunoreactive PTH(PTH 53-84) was examined in 13 patients on chronic hemodialysis. Thirteen patients were treated for 300 days (10 months), 9 patients for 520 days (14 months) and 6 patients for 720 days (2 years) with increasing doses of 1 alpha(OH)D3 intravenously under careful control of plasma Ca2+. Blood samples were obtained 1 week before start of treatment and then at every 2nd week. None of the patients had previously been treated with oral vitamin D metabolites. Intact PTH levels were maximally suppressed after 27-33 weeks of treatment by approximately 73%. At the end of the study periods, PTH 1-84 was still suppressed by 78 +/- 4.3% after 300 days, 78 +/- 8.8% after 520 days and 85 +/- 6.5% after 720 days. Plasma Ca2+ was kept within normal levels, but showed an initial increase from 1.14 +/- 0.03 to 1.27 +/- 0.15 mmol/l, and an adjustment of the doses of 1 alpha(OH)D3 was necessary. The present investigation demonstrated (1) that intravenous administration of the 1-hydroxylated vitamin D metabolite 1 alpha(OH)D3 induced a significant decrease in circulating levels of biologically active intact PTH, and (2) that it was possible to maintain the marked suppression of PTH secretion by intravenous treatment of 1 alpha (OH)D3 for up to 2 years. Hypercalcemia could be avoided by careful monitoring of plasma Ca2+ and adjustment of the doses of 1 alpha(OH)D3.


Improvement in prognosis of patients with acute renal failure over a period of 15 years: an analysis of 710 cases in a dialysis center.

In order to evaluate the changes in causes and outcome of acute renal failure (ARF) during the years 1975-1989, 710 patients treated in our dialysis center were analyzed. We compared the etiology, the severity and catabolic state of ARF, the techniques of renal replacement therapy, which were employed and the ages and mortality rates of these patients, who received dialysis therapy during the years 1975-79 (n = 227), 1980-84 (n = 240) and 1985-89 (n = 243). The number of postoperative, posttraumatic and non-traumatic cases of ARF was approximately the same in all three 5-year periods, only the frequency of postrenal failure decreased from 7% in the years 1975-79 to 3% in the years 1985-89. The incidence of sepsis as a major cause of ARF and the most important risk factor was comparably high in the surgical and medical patients during all of the periods, but it increased in the traumatic patients from 7% in the years 1975-79 to 28% during the last 5-year period. The prevalence of respiratory failure and jaundice as additional organ failures, the severity of ARF (oligonanuric-nonoliguric) and the metabolic state were not different in the three patient groups. The magnitude of rise in serum creatinine before the start of renal replacement therapy was significant lower in the last 5-year period in comparison to the years 1975-79 (p < 0.05). Hemodialysis was the treatment in choice of 98 and 93% of the cases during the first two periods, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Microalbuminuria fractional clearance and early renal permselectivity changes in essential hypertension.

In order to verify if, in essential hypertension (EH), the microalbuminuria (AER) increase could be due to hemodynamic modifications or to glomerular structural changes, in 15 essential hypertensives (EHs) with 24-hour AER > 16 micrograms/min and in 15 EHs with 24-hour AER < or = 16 micrograms/min, the day- and nighttime behavior of creatinine clearance (Ccr), as well as AER clearance (AER-C) and fractional clearance (AER-FC), and behavior of blood pressure (BP) was evaluated. Patients with 24-hour AER > 16 micrograms/min showed significantly higher values of 24-hour and daytime Ccr than the other group of EHs, while during the night period, there were no significant differences between the two groups. On the contrary, AER and both AER-C and AER-FC resulted markedly and significantly higher in the EHs with 24-hour AER > 16 micrograms/min not only in the 24-hour evaluation, but also during the nighttime study, notwithstanding the significant decrease in BP and in Ccr observed during the night. These data, in the absence of correlations between BP and AER-FC seem to demonstrate the existence in EHs with 24-hour AER > 16 micrograms/min of an altered glomerular permselectivity, due to changes of the glomerular membrane.


Deposition and removal of cutaneous beta 2-microglobulin.

These studies were designed to track the cutaneous deposition of beta 2-microglobulin (beta 2M) in patients on chronic hemodialysis, patients with chronic renal insufficiency and patients with successful renal transplants. Immunoperoxidase staining of skin biopsies from dialysis patients demonstrated significantly increased amounts of beta 2M compared to controls (p < 0.01). There was a strong positive correlation between the skin beta 2M content and the years of dialysis treatment. Renal transplant recipients had decreased skin content of beta 2M compared to hemodialysis patients. There was no difference in the skin beta 2M content in patients with chronic renal insufficiency not on hemodialysis and controls. No dialysis patient had amyloid in the skin by Congo red stain. We conclude that beta 2M accumulates in the skin of patients on chronic hemodialysis. This beta 2M is not in the form of amyloid. Successful renal transplantation allows for the removal of beta 2M from the skin indicating that beta 2M not in the form of amyloid can be mobilized from tissue sites.


Unsuspected morbid hypermagnesemia in elderly patients.

This study was designed to determine the incidence, etiology and consequences of severe hypermagnesemia. We retrospectively reviewed all hospital admissions over a 5-year period from 1984 to 1989 and identified 8 cases of severe hypermagnesemia (serum Mg > or = 6.0 mg/dl) due to magnesium ingestion. All but 1 patient were elderly (mean age 70 +/- 6 years). The etiology when identified was due to magnesium-containing cathartics (n = 3) or antacids (n = 3). The total amount of magnesium ingested was not excessive, but bowel disorders that may have enhanced absorption (such as active ulcer disease, gastritis, colitis, perforated viscus, massive gastric dilatation) were present in 7 of the 8 patients. Unexpectedly, only 1 had preexisting renal failure. Renal function was found to be normal in 1, only mildly to moderately impaired in 5 (creatinine < 3.6 mg/dl) and severely impaired in 2 (creatinine 7.6, 15.7 mg/dl). Clinical sequelae of hypermagnesemia were hypotension (n = 7), bradycardia (n = 2), respiratory depression (n = 3), EKG abnormalities (n = 6), depressed mental status (n = 5). Hypocalcemia (range 5.7-7.4 mg/dl) more severe than could be attributed to either hypoalbuminemia or acute renal failure was present in 7. A low anion gap (range-2 to 9) was present in 5. Most striking was the fact that despite clinical sequelae, the hypermagnesemia was unsuspected in 6 of the 8 cases. Hypermagnesemia can occur without severe renal insufficiency in association with bowel disease, particularly in elderly individuals, and may be a clinically unrecognized cause of cardiovascular dysfunction, hypocalcemia and neurologic or respiratory depression.


Aggressive ophthalmological management in diabetic end-stage renal disease: a study of 31 consecutively referred patients.

We prospectively followed the course of eye disease in patients with diabetic end-stage nephropathy from the time of initial referral by the renal unit. A total of 31 patients (62 eyes)--9 of whom had functioning renal transplants and 22 of whom were on maintenance dialysis--were consecutively evaluated for 6-24 months (mean follow-up of 18 months). Visual acuity and diabetic retinopathy stabilized or improved in all 18 eyes of the transplant patients and in 41 of 44 (93%) eyes of the dialysis patients. When first evaluated, economically useful vision (20/200 or better) was present in 11 of 18 (61%) eyes in the transplant group and in 25 of 44 (57%) eyes in the dialysis group. At the most recent evaluation, economically useful vision was present in 13 of 18 eyes (72%) in the transplant group versus 28 of 44 eyes (64%) in the dialysis group. Ambulatory vision (counting fingers or better) was present in 15 of 18 eyes (83%) in the transplant group versus 37 of 44 eyes (84%) in the dialysis group.


Acute and long-term effects of therapy with high-dose furosemide in chronic hemodialysis patients.

The effects of daily administration of 250-2,000 mg furosemide (F) were studied in patients on hemodialysis who still had residual renal function. In a short study, 10 patients (endogenous creatinine clearance 0.6-5.3 ml/min/1.73 m2) used 1,000 mg F twice daily during 7 days, and in a long-term study 13 patients (endogenous creatinine clearance 0.7-6.8 ml/min/1.73 m2) were treated during 1 year with 250-1,000 mg F orally each day. In the short study, we observed an increase in the 24-hour volume excretion with a median of 109% (p < 0.005). Urinary sodium excretion increased 210%, chloride 346% and potassium 65% when compared with the control period. In the long-term study, a marked initial rise in diuresis and electrolyte excretion was found. However, during a 1-year follow-up, a gradual decrease in response with time was found caused by progression of renal disease. There were no signs of ototoxicity. Side effects were bollous dermatosis on the limbs after exposure to sunlight during the summer (3 patients). We conclude that high-dose F is effective in patients on hemodialysis with residual urinary production. However, in the long term, the diuretic effects diminish because of progression of the underlying renal disease.


Blood lactate is reduced following successful treatment of anaemia in haemodialysis patients with recombinant human erythropoietin both at rest and after maximal exertion.

The effect of increasing haemoglobin by erythropoietin therapy on exercise capacity was investigated in 11 regular haemodialysis patients, previously transfusion dependent. Exercise work load increased from a median of 100 W (95% confidence limit, 25-135) to 120 (45-180; p < 0.05) following erythropoietin, and the duration of the exercise test from 13 (3.5-20) to 15.5 min (4-22; p < 0.05). Resting blood lactate concentration decreased from 0.8 (0.6-1.6) to 0.3 mmol/l (0.3-0.4), p < 0.05, following treatment with erythropoietin, as did blood lactate concentration at maximal exertion from 2.0 (1.0-4.1) to 1.8 mmol/l (0.5-2.8; p < 0.05). In association with the increase in haemoglobin from a median of 6 (5.1-6.8) to 11.1 g/dl (11-11.9) following erythropoietin therapy, patients were able to achieve greater exercise capacity both in terms of maximum work load and duration of exercise in association with a reduced resting arterial lactate and a similar exercise-induced lactate production. This suggests that treatment had improved muscle function in terms of lactate production and/or utilisation. This was probably due to the increase in tissue oxygen delivery, as there was an increase in the median arterial oxygen content from 79 (65-85) to 150 ml O2 (144-157) following erythropoietin treatment.


Serratia marcescens cellulitis in a patient on hemodialysis.

Serratia marcescens is an infrequent cause of cellulitis with only 5 reported cases. Four of the 5 patients were immunocompromised. Additionally, the cellulitis usually occurred at a site contiguous with a wound. We report a case of S. marcescens cellulitis in a patient with end-stage renal disease on chronic hemodialysis. The initial presentation was a soft tissue infection that progressed to septic shock. Ultimately, the patient responded to antibiotics and surgical debridement of infected tissue. This case serves as a reminder to consider infections due to gram-negative bacilli as a cause of cellulitis in immuno-compromised patients regardless of the presentation.


Profound persistent eosinophilia in a patient with spontaneous renal atheroembolic disease.

We describe an elderly women who died of renal failure secondary to spontaneous renal atheroembolic disease. The sole clinical clue to this diagnosis was a profound eosinophilia up to 19,100/mm3 and a relative eosinophil count of 80%. Renal atheroembolic disease should be a prominent consideration in any patient with both renal insufficiency and peripheral eosinophilia.


Renal failure in temporal arteritis.

We report a case of biopsy-proven temporal arteritis and polymyalgia rheumatica with improved clinical symptoms with steroid treatment but with subsequent renal failure while on steroids. Kidney biopsy showed focal segmental necrotizing glomerulonephritis with crescents and small-vessel vasculitis. Treatment with methylprednisolone and cyclophosphamide achieved normalization of renal function. We emphasize the importance of kidney biopsy because of its therapeutic implications. The previous literature concerning renal disease in temporal arteritis is discussed.


Does regressed posttransplantation Kaposi's sarcoma recur following reintroduction of immunosuppression?

We report a patient who developed generalized Kaposi's sarcoma (extensive skin and stomach lesions) 24 months after renal transplantation whilst on cyclosporin (CyA) and prednisolone. Kaposi's sarcoma disappeared completely upon withdrawal of CyA. The patient remained with a well-functioning graft and free of Kaposi's sarcoma for 36 months on prednisolone alone. CyA was reintroduced following an episode of acute rejection. Within 8 weeks, Kaposi's sarcoma reappeared on the skin at the same sites as the previously healed lesions. They completely disappeared again upon withdrawal of CyA. Azathioprine was then introduced and Kaposi's sarcoma lesions reappeared 6 months later.


Nephrotic-range proteinuria in a patient with high renin hypertension: effect of treatment with an ACE-inhibitor.

A 65-year-old man presented proteinuria in the nephrotic range that occurs in the setting of high renin hypertension. Proteinuria persisted after normalizing blood pressure by nifedipine. In contrast, treatment with an ACE-inhibitor (enalapril) resulted in the prompt resolution of the proteinuria. Interestingly, proteinuria relapsed after removing the ACE-inhibition. These observations suggest a causal relation between the overactivity of the renin-angiotensin system in this patient and his proteinuria.


Direct interpretation of dreams: typology.

The dream typology assorts dreams into three major categories: dreams whose origin is endogenous, exogenous, or relational. Dreams of the first type arise from somatic needs, feelings, and states that accompany organismic adjustments to system requirements. Dreams of the second type are initiated by kinetic and dispositional tendencies toward engagement and exploration of the outer world. And dreams of the third type derive from interpersonal dispositions to interaction and relationship with other people. Within each category, dreams may occur at different levels of complexity. The dream typology permits the integration of psychoanalytic observations about the dreams from a variety of perspectives within a common framework. Freud's view that a dream is a wish fulfillment finds its primary niche in endogenous need, wish fulfillment, and convenience dreams. Kohut's observations about self-state dreams and inner regulation (1971, 1977) are accommodated to the middle range of endogenous dreams, and Jung's individuation dreams (1930) occupy the advanced range. Similarly, Bonime's interpersonal approach to dream interpretation (1962) is encompassed by relational dreams of the middle level. In addition, types and modes of dreams that are only infrequently encountered in clinical psychoanalysis are accommodated. The dream typology suggests that different psychoanalytic theories are like the position papers that might have derived from the fabled committee of learned blind who were commissioned to determine the appearance of an elephant. Each individual got a hold on some part, but could not see the whole; so for each, the part became the whole. The psychoanalytic theorist is in exactly an analogous position because, in fact, he is blind to the extent of the unconscious and is constrained to what he can infer. What he can infer depends on cohort, client population, and how he calibrates his observations. The result has been procrustean interpretation, dissention, and a remarkable stasis in the psychoanalytic theory of the unconscious. The theory of the unconscious that arises from the method of direct interpretation reflects a differentiated inner world with variegated landscapes of images and frameworks. The derivatives of the unconscious are determined by complex decision rules, symbol systems, and syntax. Images and dreams possess a primary autonomy from the conscious mind and arise through the configural mind, which serves the construction and synthesis of experience and knowledge. The derivatives emerge out of common human nature conjoined with concrete human experience. For this reason, dreams and images appear universal.(ABSTRACT TRUNCATED AT 400 WORDS)


Voice of a hidden minority: identification and countertransference in the cross-cultural working alliance.

This paper has expanded the psychoanalytical definition of the working alliance to include the influence of culture. The cross-cultural working alliance is a therapeutic dyad that incorporates a psychotherapist and a patient of dissimilar backgrounds. Each participant enters the alliance with cultural introjects--values, attitudes, and ways of behaving that stem from ethnocultural roots. These introjects influence the intrapsychic world, shape the conscious worldview, and the perception of life experience. The influence of cultural predispositions on the psychopathologies of the patients was prominent in two case examples: a black West Indian male and a Korean-American male. The therapist also brought cultural predispositions to her work. A constructive countertransference phenomenon occurred in the working alliance that had a direct connection to its cross-cultural nature. This phenomenon is called associative identification--the conscious identification of therapist with the ego experience of patient. With the use of the associative identification, the therapist uncovered parallel memories that promoted theoretical understanding and informed therapeutic technique. Just as anthropologists claim that cultures share universal institutional patterns, it is proposed that universal cultural conflicts may also be observed in the cross-cultural working alliance. The analyst and patients in this study had two culture-conflicts in common: the longing to be seen, and the pull for disidentification. It is concluded that associative identification enriched the reparative quality of treatment, and encouraged the integration of isolated, split-off parts of the self.


The relationship of blood lactate concentrations, oxygen delivery and oxygen consumption in septic shock and the adult respiratory distress syndrome.

Tissue hypoxia is thought to be pivotal to the development of multiple organ failure, but cannot be measured directly in clinical practice. We assessed the relationship between initial arterial blood lactate concentrations and the presence of the phenomenon of delivery-dependent oxygen consumption, both of which may indicate tissue hypoxia. Twenty-three critically ill patients with septic shock and adult respiratory distress syndrome were studied prospectively and allocated to one of two groups according to blood lactate concentrations. In group 1, blood lactate concentration was less than the level widely accepted as significant (2 mmol.l-1); in group 2, the concentration exceeded 2 mmol.l-1. In both groups, resuscitation with colloid, blood and vasoactive drugs resulted in significant increases in oxygen delivery; in group 1 (n = 13), mean (SEM) oxygen delivery increased from 484 (36) to 730 (44) ml.min-1.m-2 (p < 0.005) and in group 2 (n = 10) from 550 (54) to 780 (54) ml.min-1.m-2 (p < 0.05). In neither group was there a significant change in oxygen consumption. However, there were individuals in both groups who exhibited pathological delivery dependence. This suggests that the absence of hyperlactataemia does not preclude delivery dependence of oxygen consumption with the attendant potential for tissue hypoxia.


Cardiovascular effects of forces applied during laryngoscopy. The importance of tracheal intubation.

The relationship between the forces applied during laryngoscopy and cardiovascular changes were studied in patients undergoing laryngoscopy with or without intubation. This enabled us to differentiate between the cardiovascular effects of laryngoscopy and the effects of tracheal intubation. The forces applied during laryngoscopy were only weakly related to the cardiovascular changes, whereas tracheal intubation had a major influence. The many difficulties encountered in interpreting results from these studies are discussed. It is concluded that tracheal intubation causes more cardiovascular changes than laryngoscopy in routine uncomplicated procedures.


Cardiovascular effects of fibreoptic oral intubation. A comparison of a total intravenous and a balanced volatile technique.

The cardiovascular response to fiberoptic oral intubation under total intravenous anaesthesia with propofol or a balanced volatile technique with thiopentone/enflurane was compared in 50 patients of physical status ASA 1 and 2 who were scheduled for elective ear, nose and throat surgery. Patients were randomly assigned to receive propofol or enflurane. There was no significant difference between the two anaesthetic techniques in haemodynamic profile either before, during or after fibreoptic intubation (the study design was adequate to detect a 20% difference with > 90% statistical power), in incidence of postoperative sore throat or in time taken for intubation. In no patient did the oxygen saturation decrease to below 95% or the CO2 tension exceed 5.8 kPa.


Patient-controlled epidural analgesia following post-traumatic pelvic reconstruction. A comparison with continuous epidural analgesia.

A randomised, single-blinded study was conducted to compare patient-controlled epidural analgesia with continuous infusion epidural analgesia for the treatment of pain following post-traumatic pelvic reconstruction. The patient-controlled group (n = 11) received a background infusion of 4 ml.h-1 of bupivacaine 0.125% with fentanyl 1 microgram.ml-1, and 3-6 ml bolus doses, self administered, as required (with a 15 min lockout interval). The continuous infusion group (n = 12) received a continuous infusion of the same solution through an identical apparatus, but with the demand button deactivated. This was started at 10 ml.h-1 and adjusted by the anaesthetist, as required, up to a maximum of 25 ml.h-1. Pain scores, side effects, and the volumes of drug infused were recorded over the first 3 postoperative days. One patient from each group was withdrawn because of catheter-related problems. Pain scores were similar and the incidence of nausea and pruritus was low in both groups. There was no recorded instance of respiratory depression or hypotension and there was no significant difference between the groups in the volumes of drug solution received. Patient satisfaction was equally very good in both groups. Patient-controlled epidural analgesia is an effective means of providing pain relief after post-traumatic pelvic reconstruction, but did not significantly reduce analgesic requirements in comparison with continuous infusion epidural analgesia.


Continuous extradural infusion of lignocaine 0.75% vs bupivacaine 0.125% in primiparae: quality of analgesia and influence on labour.

We studied 86 primiparous women with uncomplicated pregnancy and labour requesting extradural analgesia in labour. All the women were over 36 weeks of gestation with a cephalic-presenting singleton fetus. The women were allocated randomly to two groups: group A, who received an extradural infusion of lignocaine 0.75%, after an initial dose of 10 ml of lignocaine 1.5%, and group B, who received an infusion of bupivacaine 0.125% after an initial dose of 10 ml of bupivacaine 0.25%. All the women had their labour actively managed. Assessment of analgesia during labour and delivery, and the requirements for additional top-ups were noted, as were mode of delivery, requirement for oxytocic augmentation and incidence of fetal distress. Maternal and umbilical cord plasma concentrations of lignocaine were measured at delivery in 12 women receiving extradural lignocaine. There were no statistically significant differences between the two groups in terms of the mode of delivery, incidence of fetal distress, fetal heart rate abnormalities, or Apgar scores of the babies. Women in the bupivacaine group had a significantly better quality of analgesia during both the first and second stages of labour (p = 0.0005) and required fewer top-ups than those in the lignocaine group. However, the requirement for oxytocin augmentation during the first and second stages of labour was significantly less in the lignocaine group (p = 0.004). Similarly, the duration of the second stage was shorter compared with the bupivacaine group. In spite of high plasma concentrations of lignocaine, no side effects were noted in either mothers or babies.


An assessment of an alternative method of internal jugular vein catheterisation.

This study assessed a tried, but previously unpublished, method of internal jugular vein cannulation in 50 patients scheduled for elective cardiac surgery. The method involves using two landmarks: the larynx (thyroid and cricoid cartilages) together with the carotid artery (if it is palpable). The cricoid and thyroid cartilages were readily identifiable in 48 (96%) patients. The carotid artery was palpable in 45 (90%) patients, although some difficulty in palpation was recorded in 24 (48%) patients. There was a 98% success rate of venous cannulation. In 90% of cases the 21 gauge seeker needle entered the vein on the first or second attempt, and in 82% of cases the subsequent 18 gauge introducing needle entered the vein on the first attempt.


The caudal boundary of the thoracic paravertebral space. A study in human cadavers.

The caudal limit of the thoracic paravertebral space was examined in 13 human cadavers. The origin of the psoas major muscle was found to completely seal off the paravertebral space below the level of the 12th thoracic vertebra. The possibility of caudal spread of a thoracic paravertebral block below this level through the paravertebral space would appear unlikely.


Phantom limb pain during labour.

We report the occurrence of severe phantom limb pain during labour. The patient, a 27-year-old, had had an above knee amputation performed 6 years earlier following a road traffic accident but had no previous history of phantom limb phenomena. However, during early labour, she complained of a severe phantom limb pain in her amputated leg; a continuous epidural block relieved her of the sensation and pain. The sensation did not return following delivery.


Ventricular fibrillation after accidental injection of bupivacaine into the pericardium.

A postoperative cardiac surgical patient developed ventricular fibrillation immediately after accidental pericardial injection of bupivacaine at room temperature. The possible causes, which include systemic toxicity, local vasoconstriction with myocardial ischaemia, local toxic effect of bupivacaine or local hypothermia, are discussed.


Are electrocardiogram changes the first sign of impending peri-operative pneumothorax?

A patient in the right lateral position underwent left nephrectomy, after which he was placed supine for insertion of an arteriovenous fistula. All haemodynamic and respiratory values, including peak inspiratory pressure, were within normal limits and unchanged from baseline measurements. However, following the position change we noted that the amplitude of the electrocardiogram complexes were dramatically reduced. Our differential diagnosis included the possibility of a pneumothorax, which was subsequently confirmed by both physical examination and chest X ray. A chest drain was planned to be inserted at the end of the surgery, but 25 min after the electrocardiogram changes were noted, the patient's vital signs suddenly deteriorated. Emergency treatment for pneumothorax was instituted with good effect. The diagnostic use of the electrocardiogram and the treatment of this intra-operative pneumothorax are discussed.


Anaesthesia for thoracoscopic pleurectomy and ligation of bullae.

A patient is described who, despite severe pre-operative respiratory disability, had her persistent pneumothorax successfully managed by thoracoscopic pleurectomy. The technique causes considerably less pain and interference with respiratory function postoperatively than does conventional thoracotomy. Potential anaesthetic problems arise because of the necessity of insufflating carbon dioxide at pressures of up to 1 kPa to maintain a pneumothorax during surgery.


Transoesophageal echocardiography during removal of a caval filter in a patient at high risk of massive pulmonary thromboembolism.

A new type of vena caval filter was to be removed from a woman who had a high risk of massive pulmonary thromboembolism because of extensive thrombus in the iliac vein incorporating the filter. The removal was performed under general anaesthesia in the cardiac operating theatre using transoesophageal echocardiography to monitor the right heart and the pulmonary artery during the critical phase of removal. The manoeuvre succeeded with only insignificant embolisation occurring, and that was identified by transoesophageal echocardiography.


The use of a checklist for anaesthetic machines.

The use of the Association of Anaesthetists of Great Britain and Ireland checklist for anaesthetic machines, based on an oxygen analyser, was surveyed over a 5-week period in a teaching hospital. Fifty-five completed checklists were analysed; no problems developed during anaesthesia which were missed by the checklist. The mean time taken to complete the checklist for one machine was 8.9 min with a range of 5 to 19 min; for two consecutive machines it was 18.25 min with a range of 10 to 30 min. The most frequent faults detected were the poor reliability of some oxygen analysers, absent ventilator disconnection alarms, and absent oxygen supply failure alarms on some older machines. Faults were found in 60% of the machines checked; 18% of these were deemed to be serious.


A comparison of the performance of two types of infusion device.

The delivery performance of two types of infusion pump, the IVAC 711 series syringe driver and the IVAC 531 series drop-counter, was examined by measuring the output of 16 pumps. We found that despite careful attention to the setting up of all apparatus, the IVAC 711 series syringe drivers had a significant lag phase before output matched the set rate, and that this lag phase was more pronounced at low rates of infusion. This was not the case with the IVAC 531 series which delivered the set rate from the outset. We suggest, that when the timing and rate of an infusion of drug is important from the outset and when these types of pump are being used, that either the syringe driver pump is primed by a brief period of high rate infusion vented by a three-way tap or that a drop-counter pump is used.


An evaluation of the behaviour of children undergoing dental extraction under general anaesthesia.

The cooperation of a random sample of 190 children with an average age of 5 years (range 1-17 years), presenting with a dental emergency requiring tooth extraction under general anaesthesia, was evaluated using the Frankl Behaviour Scale. The results showed that 60% of the children cooperated in the operating theatre. Furthermore, removal of only one or two teeth per patient accounted for 40% of the cases. This survey has identified a group of child dental patients who might benefit from conscious sedation and local anaesthetic techniques.


Diclofenac for analgesia after caesarean section.

The analgesic efficacy of a single intramuscular dose of 75 mg diclofenac given after elective Caesarean section was studied in 50 women in a double-blind randomised manner using a patient-controlled analgesia system. The mean 18 h papavaretum consumption of the placebo group was significantly greater (91.4 mg compared to 61.4 mg). Subjective experience of pain and observed sedation were significantly greater in the control group up to 6 h after operation.


The effect of paracetamol or diclofenac administered before operation on postoperative pain and behaviour after adenoidectomy in small children.

We compared the effects of rectally administered diclofenac (12.5 mg) with paracetamol (125 mg) on pre- and postoperative behaviour and the need for supplementary analgesia in 44 children scheduled for adenoidectomy (with or without myringotomy). The study drugs were given in combination with diazepam (0.5 mg.kg-1) about 20 min before the children were taken to the operating theatre. On arrival there, the children who had received diclofenac were significantly quieter (< 0.05), easier to handle (p < 0.01) and cried less (p < 0.05) than those in the paracetamol group. During recovery, children in the diclofenac group needed fewer supplementary doses of intravenous pethidine than those receiving paracetamol (p < 0.001). There were no obvious differences between the groups in intra-operative bleeding (as estimated by the surgeon), or in measured blood loss. No postoperative complications became evident. The pre-operative rectal administration of diclofenac for pain relief after adenotomy is safe and effective.


EMLA for postoperative analgesia for day case circumcision in children. A comparison with dorsal nerve of penis block.

A randomised study was designed to evaluate and compare the analgesic efficacy of EMLA with dorsal nerve of penis block, in children undergoing day case circumcision. The mean (SD) duration of analgesia for EMLA and dorsal nerve block were 0.5 (0.1) h and 6.4 (1.2) h respectively (p < 0.001). It is concluded that EMLA is not as effective as dorsal nerve of penis block with regard to postoperative analgesia. No adverse effects were observed.


A study into the incidence of methaemoglobinaemia after 'three-in-one' block with prilocaine.

A 34-year-old female patient developed methaemoglobinaemia following a femoral nerve block using prilocaine. The concentrations of methaemoglobin in the blood of the next eight patients receiving this block were assayed, and found to be significantly increased above baseline concentrations, but very significantly less than the concentration in the patient with symptomatic methaemoglobinaemia. Pulse oximetry was of value in making the diagnosis.


Administration of vancomycin during cardiopulmonary bypass.

Vancomycin was given during cardiopulmonary bypass to 12 anaesthetised patients undergoing open heart surgery. Injection of vancomycin 1 g within 60 s via the venous inlet of the oxygenator resulted in a moderate and transient decrease of mean arterial pressure. This minimal reaction may be attributed to dilution of vancomycin by the extracorporeal circuit volume, to the bypassing of the lungs which are a major site of storage of vasoactive substances, or to the maintenance of adequate perfusion flow during cardiopulmonary bypass. The results suggest that the haemodynamic adverse reactions to vancomycin, given as antibiotic prophylaxis, may be decreased by its administration after initiation of cardiopulmonary bypass.


Intermittent positive pressure ventilation through a laryngeal mask airway. Is a nasogastric tube useful?

A nasogastric tube was used to aspirate air insufflated into the stomach during intermittent positive pressure ventilation through a laryngeal mask airway and a tracheal tube. No difference was found in the amount aspirated between patients with a tracheal tube, a laryngeal mask airway with the nasogastric tube closed or a laryngeal mask airway with the nasogastric tube open, when the nasogastric tube was aspirated at 15 min intervals for the first hour of anaesthesia.


Postoperative oxygenation in the elderly following general or local anaesthesia for ophthalmic surgery.

Peripheral oxygen saturation was recorded on the pre-operative night (between 2400 and 0600 h), the immediate postoperative period (first 60 min) and the first postoperative night (2400 to 0600 h) in 18 elderly patients aged 70 years or over presenting for elective ophthalmic surgery. Nine patients had surgery performed under general anaesthesia employing muscle relaxants and controlled ventilation and nine under local anaesthesia using a peribulbar block. The median (interquartile range) percentage of time during which the patients had an oxygen saturation of less than 90% was 0 (0-0.2) and 0.04 (0-0.4) on the pre-operative night, 0.7 (0-1.4) and 0.3 (0-1.2) in the immediate postoperative period, and 0.05 (0-0.16) and 0 (0-0.3) on the postoperative night in the general and local anaesthesia patients respectively. There were no significant differences between general and local anaesthesia in respect of these data and the overall incidence of significant desaturation was low. The present study could not demonstrate any adverse effect of general anaesthesia on oxygen saturation in patients undergoing minimally invasive surgery.


[The concentration of atrial natriuretic peptides (ANP). ANP in different sections of the circulation during atrial volume load with and without anesthesia].

We studied the effect of a volume load induced by a 45 degrees Trendelenburg position on atrial natriuretic peptide (ANP) secretion in awake and anaesthetized patients with coronary artery disease undergoing aortocoronary bypass surgery. ANP was measured in different parts of the circulation before and after induction of high dose fentanyl anaesthesia at fixed times prior to and after extracorporeal circulation. METHOD. In eight patients with coronary artery disease (NYHA classification II-III), who received neither diuretic nor positive inotropic therapy, ANP was measured in the various parts of the circulation: in a peripheral vein, a radial artery, in the pulmonary artery and in the coronary sinus. The measurements were made in the supine and 45 degrees Trendelenburg position. Measurements of mean arterial pressure (MAP), central venous pressure (RAP), pulmonary arterial pressure (PAP), pulmonary capillary wedge pressure (PCWP), cardiac index (CI) and heart rate (HR) were taken simultaneously. The measurements were taken in the awake patient, during steady-state high-dose fentanyl anaesthesia with 50% O2 in N2O and after extracorporeal circulation. RESULTS. Compared to measurements in a control group, ANP levels were significantly higher in all parts of the circulation in patients with coronary artery disease, although clinical symptoms of heart failure were absent. After extracorporeal circulation, significantly higher levels of ANP were found at all measurement sites; however the concentration gradient of ANP between coronary sinus and arterial or venous blood was reduced. In awake and anaesthetized patients a change in body position, causing a significant increase in filling pressures, did not produce an increase in ANP levels at all measurement sites. The induction of high-dose fentanyl anaesthesia did not have an influence on plasmatic ANP levels. CONCLUSION. The results of this study lead to the following conclusions: 1. ANP levels in patients with CAD are increased, even if clinical heart failure symptoms are absent. 2. ANP is secreted in the coronary vessels. Following dilution in the atrial blood, it is metabolized to inactive compounds in the periphery. 3. Basic ANP levels are not changed by high-dose fentanyl anaesthesia. Marked increases of the filling pressures do not correlate with atrial ANP levels either before or after induction of anaesthesia. 4. After extracorporeal circulation ANP levels are significantly increased in all parts of the circulation. The concentration gradient between coronary sinus blood, on the one hand, and arterial and venous blood on the other hand is reduced. This phenomenon is probably caused by an alteration in the metabolism of ANP during hypothermic extracorporeal circulation.


[Hemodynamics of coronary surgery patients following magnesium aspartate infusion].

Hypertension is a common phenomenon in patients undergoing aortocoronary bypass grafting. This hypertension increases myocardial oxygen consumption and can be prevented by application of vasodilators. A possible cause is activation of the renin angiotensin system. Magnesium is a potent vasodilator and has a beneficial effect after myocardial ischaemia. The study was performed to analyse the influence of magnesium infusion on the haemodynamic status and plasma renin activity in patients undergoing aortocoronary bypass grafting. METHODS. Eighteen patients (NYHA classification II-III) undergoing bypass surgery were divided into two groups, a magnesium and a control group. The magnesium group (n = 9) received 0.8 mEq/kg per h magnesium aspartate as an infusion for 15 min while still awake. After induction of anaesthesia, the magnesium infusion was reduced to 0.2 mEq/kg per h and stopped after aortic cannulation was completed. Plasma magnesium levels and concentrations within erythrocytes were measured. Anaesthesia was induced by flunitrazepam (0.01 mg/kg), fentanyl (0.005 mg/kg) and pancuronium (0.1 mg/kg). After intubation, patients were normoventilated with N2O/O2 = 1:1 and isoflurane (0.5-1.0 vol%). Additional doses of fentanyl (0.0025 mg/kg) were injected before the incision and before sternotomy. Mean arterial pressure, heart rate, cardiac index, total peripheral resistance, pulmonary vascular resistance, mean pulmonary arterial pressure, pulmonary capillary wedge pressure, left ventricular stroke work index, right ventricular stroke work index, intrapulmonary shunt and plasma renin activity were evaluated at five predefined points: (1) prior to magnesium infusion; (2) after magnesium infusion; (3) 10 min following induction of anaesthesia under steady-state conditions; (4) after sternotomy; (5) after aortic cannulation. RESULTS. Concerning the haemodynamic parameters (MAP, RAP, PAP, PCWP) no significant difference between the two groups could be demonstrated. In the control group peripheral resistance (TPR) was higher following sternotomy and aortic cannulation than in the magnesium group. Magnesium prevented decrease of the cardiac index (CI) under steady-state conditions, during sternotomy and following aortic cannulation. Left and right ventricular stroke work indexes (LVSWI and RVSWI) were higher in the magnesium group. Plasma renin levels were not significantly different between the two groups. CONCLUSION. Patients undergoing cardiac surgery benefit from magnesium administration in the pre-bypass phase. Due to its vasodilating effect, magnesium lowers the output impedance of the left ventricle and improves cardiac pumping function. It opposes detrimental cardiovascular responses to sternotomy and following aortic cannulation. Also of importance is the advantageous effect of magnesium on cardiac arrest elicited by cardioplegia and for reactivation of the ischaemic myocardium.


[Interactions between lithium/rubidium and six muscle relaxants. A study on the rat phrenic nerve-hemidiaphragm preparation].

The cumulative concentration-response curves for alcuronium, metocurine, d-tubocurarine, suxamethonium, pancuronium and vecuronium alone or in combination with lithium or rubidium were measured using indirectly evoked twitch tension in the isolated rat phrenic nerve-hemidiaphragm preparation. In the experiments with lithium and rubidium, 10% and 2% of the sodium ions (118 mmol/l) of Krebs solution were replaced by lithium (11.8 mmol/l = 0.082 g/l) or rubidium (2.36 mmol/l = 0.202 g/l) ions, respectively. At these concentrations, neither lithium nor rubidium alone affected twitch tension. Lithium led to a statistically significant enhancement of the myoneural blocking effects of suxamethonium, pancuronium and vecuronium, but not of the other myoneural blocking drugs. Rubidium behaved similarly to lithium, except that enhancement of the effect of vecuronium was not statistically significant. At higher concentrations, lithium and rubidium alone depressed twitch tension in a dose-dependent manner. Neither myoneural blocking drugs nor lithium and rubidium affected the directly evoked twitch tension. It is concluded that myoneural blocking drugs, especially the newer ones, should be used with caution in patients receiving lithium or other alkali metal therapy as it is not known what interactions may arise.


[Pediatric surgery. A comparison of spinal anesthesia and general anesthesia].

Forty patients aged 2 to 5 years who were admitted for paediatric operations were randomly assigned to have either spinal or general anaesthesia. Spinal anaesthesia was achieved with isobaric bupivacaine 0.5% at a dose of 0.5 mg/kg. General anaesthesia was induced with thiopentone 2-5 mg/kg and continued with low-dose fentanyl (1-2 micrograms/kg, oxygen/nitrous oxide/isoflurane (30/70/0.1-0.5%), vecuronium normoventilating the patients. The time spent in the operation room was shorter in the spinal anaesthesia group because the children were awake and could immediately be transferred. The haemodynamic pattern and respiratory function were stable during spinal anaesthesia. After general anaesthesia, respiratory function deteriorated as indicated by arterial desaturation (< 90%), which was detected in 11 of the 20 patients after general anaesthesia. Vomiting (2), sore throat (4) and micturition difficulties (2) were the adverse events associated with general anaesthesia. Three patients were restless after spinal anaesthesia. It can be concluded that spinal anaesthesia is a suitable anaesthetic technique for paediatric surgery.


[The reuse of 29-gauge spinal needles following combined spinal-epidural anesthesia].

The technique of combined spinal epidural anaesthesia (CSE) combines the versatility of spinal with the variability of epidural anaesthesia. Spinal application of the local anaesthetic achieves a fast response, reliable sensorial and motor block at a low dose with little toxicity. The epidural catheter allows for the duration of surgical anaesthesia to be extended and provides analgesia for the postoperative period. As the incidence of post dural puncture headache (PDPH) is inversely related to the size of the spinal needle, PDPH rarely or never occurs when 29 gauge needles are used. In 1775 parturients receiving spinal anaesthesia for caesarean section, Dittmann et al. [4] reported an incidence of PDPH of 1.37% with 29 gauge needles. The 29 gauge needle produced by Becton-Dickinson is the one now most frequently used for this technique. It is recommended that these needles be reused after resterilization. The objective of this study was to examine how clean 29 gauge spinal needles really are after resterilization. MATERIALS AND METHODS. Fifteen needles (29 gauge; Becton-Dickinson) were routinely used for combined spinal epidural anaesthesia. After identification of the epidural space at the L3-4 spinal segment with the 18 gauge Tuohy needle, the 29 gauge needle was advanced through the Tuohy needle. Immediately after use the needles were cleaned, rinsed with 20 ml distilled water, dried with pressurized air and subsequently resterilized in gas. Preparation of the used needles was in accordance with the manufacturer's recommendations ("wash, rinse, dry, sterilize before initial and each subsequent use") and with generally accepted principles [6]. Two needles were additionally cleaned in an ultrasonic bath for 15 min. The needles were examined using a scanning electron microscope. After this analyses, eight needles were sterilized again and then taken for hygienic examination. They were incubated with trypticase soy broth and checked for bacterial growth. RESULTS. Scanning electron microscopy (ScEM) showed organic impurities on all needles. These impurities were equally distributed among all needles. Even the two cleaned in the ultrasonic bath were not free of organic particles. However, no material defects or damage could be seen. Hygienic evaluation proved sterility as no bacterial growth could be detected. CONCLUSION. Owing to the possibility of medico-legal consequences, which sometimes occur a long time after anaesthesia has been given, we think it is unwise to reuse such needles. We hope that disposable and cheap 29 gauge needles will soon become available.


[Fiberoptic determination of the position of the laryngeal mask].

The laryngeal mask airway (LMA) provides a patent airway when placed 'blindly' into the hypopharynx. At the laryngeal side it is supposed to form a seal surrounding the laryngeal inlet with the epiglottis lying outside the mask aperture. This study is designed to assess the prelaryngeal position of the mask by the fibreoptic technique. METHODS. After approval by the local ethical committee and informed consent, 100 adult patients (ASA groups I and II) undergoing general anaesthesia for extracorporal stroke wave lithotripsy (ESWL, Lithotripter HM 3, Dornier) of the kidney were studied. Anaesthesia was induced with propofol (1.5-2.5 mg.kg-1) and fentanyl (1-1.5 micrograms.kg-1) and maintained with isoflurane and N2O (65% in O2) as clinically indicated. The LMA was left in situ until the patients opened their mouth on command. Monitoring consisted of an ECG (SMV 104-D, Dornier), a pulse oximeter (Nellcor 200, Draeger), and a non-invasive blood pressure monitor (BP 103 N, Hoyer). Clinical assessment of airway patency and fibreoptic laryngoscopy (BF Typ 10, Olympus)--immediately and 20 min following the insertion of the LMA--were performed by two observers. RESULTS. The insertion of the LMA was successful on the first attempt in 89 patients while 5% required two, 4% three and 2% four attempts. 'Blindly' inserted without neuromuscular blockade the LMA provided a clinically sufficient airway in all patients. A central position of the LMA was assessed in only 59% of the cases. In 4 patients the mask was riding on the vocal folds. Positioned at the posterior larynx the cuff produced a compression of the laryngeal orifice when insufflated. Oblique insertion of the LMA or oblique head position during insertion produced a misplacement of the LMA. In 5 cases the LMA followed lateral movements of the head without losing its central position. In 87% the epiglottis was within the lumen of the LMA. Secretions inside the mask lumen or at the anatomic structures were seen in 36%. During manual ventilation with high inspiratory pressure (> 25 cm H2O) the oesophagus opened in 10 cases. CONCLUSIONS. Previous studies have suggested that the LMA takes a 'perfect' position at the laryngeal side when a clinically patent airway is recognized. In contrast, our results demonstrated that a central position of the LMA is achieved in only 59% of the cases. Our results indicate that epiglottic downfolding or left/right side or anterior/posterior misplacement are common but generally provide a satisfactory patent airway. This is consistent with fibreoptic findings in children and radiological observations in adults. The LMA is an essential enrichment to conventional airway management. It provides a better seal than the face mask, especially in bearded or in old patients where the facial contours are often not suited to the mask. Ideal indications seem to be elective operations of intermediate duration (1-2 h). The LMA does not protect against aspiration. For patients who are at risk of regurgitation of gastric contents, use of the LMA is absolutely contraindicated. Relative contraindications are local pathology of the pharynx and situations with low pulmonary compliance and/or high airway resistance (massive obesity, asthma, etc.), especially during controlled ventilation. Further studies are necessary to establish definite indications for the application of the LMA.


[A study of the parameters of the delivered tidal volume. Ventilation on a lung model using the CICERO anesthetic ventilator].

In many anaesthesia ventilators in common use, the tidal volume delivered is different from the tidal volume preset on the respirator. Tidal volume delivered by mechanical ventilation during anaesthesia may be influenced by fresh gas flow (FGF), the respiratory rate (RR) or the inspiratory: expiratory ratio (I:E). This may cause inadequate hypo- or hyperventilation in small children, especially in newborns and neonates. Using small tidal volumes from 20 to 100 ml preset on the respirator, we investigated in a lung model the tidal volumes delivered by the anaesthesia ventilator CICERO (Dräger, FRG) with variations of FGF, RR and I:E. MATERIAL AND METHODS. The anaesthesia ventilator CICERO (software version 4.16) was equipped with the low-compliance tubes of the "Ulmer Kinder-Set" (Rüsch Co.) and the regular CO2 canister (1500 ml) of the machine. The circuit was connected to a lung model consisting of a glass clyinder filled with copper wool with a compliance of 3.3 ml/mbar. To create a pressure-volume correlation of the entire system, i.e. the lung model, the anaesthesia circuit and the ventilator, calibrated glass syringes were used and the pressure increase in the test lung was measured. This pressure-volume correlation was linear. The pressure increase in the lung model caused by the tidal volume during ventilation therefore reflected the actual tidal volume delivered. The study was performed with small tidal volumes from 20 to 100 ml that could be adjusted exactly on the ventilator. Delivered tidal volumes were studied by varying the FGF from 1 to 6 l/min and the RR from 20 to 60/min (with I:E = 1:1.5) and by varying the RR from 20 to 60/min and the I:E from 2:1 to 1:3 (with FGF = 21/min). RESULTS. By varying FGF, RR and I:E no changes in delivered tidal volumes were noted. In all settings of the ventilator studied, the delivered tidal volume was similar to the desired tidal volume preset on the ventilator. The highest deviation from the delivered tidal volume to the tidal volume preset was 17.5% with a tidal volume of 20 ml. In preset tidal volumes 30-100 ml this deviation was lower than 10%. An intermittent "auto-PEEP" up to 5 mbar was noted during high respiratory rates (50 and 60/min) combined with an I:E at 2:1 and 1:1 or with a FGF at 4 or 6 l/min. The compliance of the ventilator equipped with the circuit was 4.2 ml/mbar. CONCLUSION. The findings in this study prove that with tidal volumes ranging from 20 to 100 ml the actual tidal volume delivered by the anaesthesia ventilator CICERO is equivalent to the tidal volume set on the machine regardless of the variation of FGF, RR and I:E. These findings are mainly based on two circumstances. Firstly, fresh gas flow is fed into a reservoir and not added to the volume delivered by the bellow during inspiration as in many other respirators. Secondly, the CICERO works with a compliance correction function integrated into the machine. Computed compressible volume from the circuit and the ventilator is added to the tidal volume preset on the ventilator; therefore, the volume delivered by the bellow consists of the volume set on the ventilator plus the compressible volume. With these characteristics the anaesthesia ventilator CICERO meets important requirements for a ventilator in paediatric anaesthesia. However, for final assessment further clinical studies are required.


[Analgesia and sedation in intensive care patients].

Analgesia and sedation with the associated reduction of undesired vegetative reactions are important components in the therapeutic regimen of intensive care patients. None of the sedative drugs available can fulfil every one of the criteria expected of an "ideal" sedative. Four commonly used drug combinations have been established as standards: 1. opioid and neuroleptic, 2. opioid and benzodiazepine, 3. ketamine and benzodiazepine, and 4. opioid and propofol. In everyday use one must take not only the specific side-effects of a drug into consideration but also its pharmacokinetic properties. These are often markedly altered in critically ill patients who have impaired functions of vital organs. The pharmacokinetics of a drug is affected by disturbed renal or hepatic function, interactions with other drugs, altered protein binding and the induction or inhibition of metabolic enzymes. The best method of drug administration is by motor-driven pump, with which large fluctuations of the dosage can be avoided. Constant ratios of drug combinations are not recommended, since the pharmacokinetics of each drug is affected to a different degree in the critically ill patient. Withdrawal symptoms, can occur for example after prolonged administration of benzodiazepines, can often be avoided by slowly reducing the dose or by switching to a short-acting substance. In some patients (e.g. those with a history of alcohol abuse) a massive increase of the drug dose is not indicated when the effect is not adequate. Instead, an entirely different substance should be employed and the administration of less frequently used drugs should be considered. Despite detailed knowledge of the altered pharmacokinetics in critically ill patients, the drugs should be dosed as dictated by the situation, true to the anaesthesiologists' adage: "Dosage according to effect!"


A preparation and purification of [1-14C]acetylcarnitine.

[1-14C]Acetylcarnitine was prepared from [1-14C]acetate and L-carnitine using acetyl-CoA synthetase and carnitine acetyltransferase. The product was purified by ion-exchange and thin-layer chromatography. Conversion of [1-14C]acetate to [1-14C]acetylcarnitine was better than 90% and overall recovery of the pure product was greater that 80%.


Temperature-dependent aggregation of pH-sensitive phosphatidyl ethanolamine-oleic acid-cholesterol liposomes as measured by fluorescent spectroscopy.

pH-sensitive liposomes made of phosphatidyl ethanolamine-oleic acid-cholesterol (4:2:4 molar ratio) at neutral pH values aggregate at approximately 40 degrees C. The aggregation is accompanied by liposome destabilization and by the release of intraliposomal fluorescent marker (calcein). Both aggregation and calcein leakage start at the temperature corresponding to the lipid phase transition into hexagonal phase. In the system studied the phase transition temperature interval is within 45 to 55 degrees C as estimated with the use of the fluorescent probe 1,6-diphenylhexatriene. The presence of cell cultivation medium RPMI 1640 decreases liposome aggregation temperature. The addition of 10% serum to the system decreases the temperature at which the aggregation proceeds still further. The conclusion that serum-free media should be used for cell experiments involving pH-sensitive liposomes is made.


Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes.

The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins. Some variability persisted even when optimized electroblotting procedures were used which reduce the sodium dodecyl sulfate (SDS) concentration in the gel and improve protein-PVDF binding. The membranes which were evaluated could be grouped as either "high retention" membranes (ProBlott, Trans-Blot, and Immobilon-PSQ) or "low retention" membranes (Immobilon-P and Westran). The high retention membranes showed higher protein recoveries under most conditions tested, especially for small proteins or peptides. These high retention membranes were also less sensitive to the exact electroblotting conditions, especially those factors which affect the amount of SDS present during either electrotransfer or direct adsorption from protein solutions. High retention PVDF membranes are therefore preferred in most cases for optimal protein or peptide recovery prior to direct sequence analysis. In contrast, low retention membranes are preferred for procedures where subsequent extraction of the proteins from the membranes is required. Even under identical conditions, substantial protein-to-protein variation for both adsorption and subsequent extraction is routinely observed for both groups of membranes, indicating that the nature of protein-PVDF interactions is more complex than simple hydrophobic interactions.


Cycled DNA immunoprecipitation procedure to enrich the target sequences for DNA binding proteins with the fold purification monitored.

Using centromere DNA binding protein (CENP-B) expressed as a fusion to beta-galactosidase in Escherichia coli, we established a cycled DNA immunoprecipitation procedure for enriching CENP-B binding sequences and monitoring the enrichment process. Degenerated synthetic oligonucleotides for an authentic CENP-B binding sequence, inserted into a pUC-derived vector, were incubated with the crude CENP-B extract. DNA-protein complexes formed in vitro were immunologically precipitated utilizing the beta-galactosidase moiety as a tagged antigen. The effectiveness of repeating cycles of immunoprecipitation was demonstrated by the color selection method designed for pUC-derived plasmids, after introducing the precipitated plasmids into Escherichia coli. After three cycles of DNA immunoprecipitation, only a few kinds of sequences constituted the majority. By repeating two more cycles, the most predominant sequence was finally enriched until homogeneous, indicating the enrichment of the binding sequences in a hierarchical order. Further application to human genomic DNA showed that two EcoRI DNA fragments, 0.49 and 0.78 kb in size, were exclusively identified. This procedure can be applied to the systematic analysis of binding sequences for any other DNA binding proteins without production of any specific antibodies or further purification.


The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.

A procedure using 2,4,6-trinitrobenzenesulfonic acid (TNBS) for the determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials is presented. The major modification from previous procedures is an extended TNBS reaction time to allow a stoichiometric reaction with amino groups. In addition, autoclave hydrolysis is used to assure sample dissolution for spectrophotometric measurements. The assay accuracy was evaluated by determining epsilon-amino groups of insulin and bovine albumin. The determinations differed from literature values by < or = 3.3%. The epsilon-amino group content of Type B gelatin was found to be 33.0 mol/gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen. The coefficient of variation for determinations on all three materials was < or = 5.3%. The assay should be applicable to a broad range of proteinaceous materials.


Fluorometric quantitation of adenosine concentration in small samples of extracellular fluid.

Adenosine is a naturally occurring nucleoside which regulates many physiological processes by interacting with adenosine-specific receptors. Knowledge of the extracellular adenosine concentration at the site of adenosine receptors on target cells is required for an understanding of mechanisms involving the action of the nucleoside. Samples of extracellular fluid which reside in close proximity to the surface of target cells are frequently small in volume. This report describes improvements in accuracy and reliability of a fluorometric assay designed for determining the concentration of adenosine in microliter samples of extracellular fluids. The utility of the assay is demonstrated by determining adenosine concentrations in interstitial and coronary effluent samples from normoxic perfused rat hearts. The assay also clearly detects changes in the interstitial and coronary effluent adenosine levels produced by isoproterenol stimulation or hypoxia. Thus, this assay is useful for determining the adenosine concentration in microliter samples of extracellular fluid and should facilitate investigations dealing with the functions of adenosine.


Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone.

An immune-tolerizing protocol was employed to generate monoclonal antibodies to a variant protein isoform of bovine growth hormone arising from alternative pre-mRNA processing. Variant bovine growth hormone used for immunization was obtained by expression in bacteria and electroelution of the protein from preparative sodium dodecyl sulfate-polyacrylamide gels. Balb/c mice were first immunized with wild-type bovine growth hormone in the presence of the cytotoxic drug cyclophosphamide, thereby tolerizing the mouse to common epitopes shared among the two proteins. Subsequently, the mice were immunized with variant bovine growth hormone to produce antibodies specific to variant epitopes. Comparisons of fusions resulting from standard and tolerizing immunization protocols resulted in a significantly enhanced production of variant bovine growth hormone-specific antibodies as a result of the immunotolerizing protocol. The specificity of the antibodies to the variant growth hormone was substantiated by differential enzyme-linked immunosorbent assay and Western blot. Nearly all hybridomas positive for variant growth hormone were negative for wild-type growth hormone. Finally, the antibodies were used to demonstrate intracytoplasmic staining of COS I cells transiently transfected with a variant growth hormone-producing plasmid. Given the power of the polymerase chain reaction to conveniently clone alternatively processed mRNA species, followed by expression in bacteria to provide antigen, the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms.


Application of Fourier transform infrared spectroscopy to studies of aqueous protein solutions.

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.


Synthesis of poly(ADP-ribose)-agarose beads: an affinity resin for studying (ADP-ribose)n-protein interactions.

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.


A quantitative assay for fragmented DNA in apoptotic cells.

Cleavage of cellular chromatin at internucleosomal sites is a characteristic change of DNA integrity in cells undergoing apoptosis. We have developed an assay for quantitation of internucleosomal DNA fragmentation in apoptotic cells. This technique involves purification of cellular DNA, dephosphorylation of the DNA ends, labeling of DNA with 32P at the 5'-end, gel electrophoresis through agarose, and quantitation of the radioactivity in DNA bands. This assay, which is about 1000- to 2000-fold more sensitive than visualization of DNA bands by ethidium staining, allows the detection of DNA fragments at picogram levels. A method for quantitatively determining the number of fragmented DNA strands is also described. Application of this new assay to evaluate the time course of internucleosomal DNA fragmentation was demonstrated in apoptotic cells induced by an anticancer nucleoside analogue.


Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography.

A fully automated method for assaying the collagen crosslinking amino acids, pyridinoline and deoxypyridinoline, in human urine samples or tissue hydrolysates is described. Samples were processed using a Gilson ASPEC system with solid-phase extraction of the crosslinks on columns containing 100 mg of microgranular cellulose. Introduction of an additional solvent step during sample preparation allowed direct analysis by reversed-phase HPLC and elimination of the drying step used previously in a manual method. Use of a synthetic pyridinoline derivative as internal standard enabled accurate quantification of the crosslinks by correcting for recoveries through the whole assay. Samples were analyzed in sequential mode with a total assay time of 30 min. The automated assay showed close correlation with the manual method for both free and total crosslink determinations in human urine (r > 0.97). Reproducibility was improved, as seen from replicate analyses of human urine (CV < 3% for automated pyridinoline measurement compared with 8-12% previously observed for the manual method). Crosslink excretion is the most useful marker of collagen degradation in metabolic bone diseases and arthritic disorders. The automated assay which has been developed is rapid, convenient, and reliable and will greatly facilitate the monitoring of urinary collagen crosslinks and their tissue levels in clinical investigations.


Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods.

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.


Fluorimetric DNA assay for cell growth estimation.

A growth assay using the fluorescent dye Hoechst 33258 has been developed which allows sensitive and rapid analysis of the DNA content of a variety of cell types including keratinocytes and mucus-secreting cells, and which requires a minimum of liquid handling. The assay can detect as few as 500 diploid human cells, and is compatible with the simultaneous detection of [3H]thymidine incorporation in the same cultures.