Solid-phase synthesis of hydroxyethylamine angiotensin analogues.

Three hydroxyethylamine analogues of angiotensins II, III, and IV were prepared by solid-phase methods. The resin-bound peptide was alkylated with the iodomethylketone derivative of the N-terminal amino acid, followed by reduction to the alcohol using sodium borohydride. The iodomethylketones can be made in good yields from commercially available N-protected amino acids. The compounds were evaluated for their ability to displace labeled angiotensins from bovine adrenal membranes, and their metabolic stability tested in kidney homogenates and aminopeptidase M preparations. The hydroxyethylamine amide bond replacement reduced the affinity of the analogues; however, they were substantially more stable to enzymatic degradation.

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function.

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT1 receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT1 receptor subtype. Pretreatment with the specific AT4 receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT1 receptor subtype.

Improved method for measuring C1-r-C1-s-(C1 inh)2 complexes by an enzyme-linked immunosorbent assay.

Measurement of C1-r-C1-s-(C1 inh)2 complexes in serum or plasma by enzyme-linked immunosorbent assay (ELISA) has been proposed as a relatively convenient and sensitive means for assessing C1 activation. However, interference by unactivated C1q (r-s)2 at low serum or plasma dilutions has resulted in estimates that vary widely with the degree of serum or plasma dilution. Precipitating the interfering C1q (r-s)2 with 6% polyethylene glycol has been proposed to resolve this problem, but here it is shown that this procedure also precipitates or coprecipitates some of the C1-r-C1-s-(C1 inh)2 complexes. Satisfactory results have been achieved without PEG precipitation by testing high plasma dilutions under conditions where there is a sufficient excess of anti-C1s coating the microtitration plate wells that removal of C1q (r-s)2 is not necessary. Optimizing conditions for quantitating these complexes at high dilution have been investigated. The mean normal EDTA plasma C1-r-C1-s-(C1 inh)2 complex measurement was 36.6 +/- 7.0 (S.D.) ELISA units with a 95% confidence interval of 19.5-47.6u. Besides providing a sensitive assay for C1 activation, measuring C1-r-C1-s-(C1 inh)2 complexes may help to clarify the pathophysiologic mechanisms resulting from C1 inh deficiency under various conditions.

Comparison of the Wang 19-gauge and 22-gauge needles in the mediastinal staging of lung cancer.

Transbronchial needle aspiration (TBNA) offers the unique opportunity to pathologically stage patients with lung cancer at the time of diagnostic bronchoscopy. The purpose of this study was to compare the staging sensitivities of the Wang 22-gauge and 19-gauge needles. We studied 64 patients with bronchogenic carcinoma and mediastinal adenopathy. Before bronchoscopy each patient underwent chest CT. Three to four aspirates were obtained with each needle from endotracheal sites adjacent to paratracheal lymphadenopathy. In 47 patients malignant mediastinal adenopathy was confirmed by the 19-gauge needle. A total of 29 patients had malignant 22-gauge needle aspirates. Of the 64 patients, 9 had benign, reactive mediastinal lymph nodes. There were 20 patients in whom only the 19-gauge needle demonstrated malignancy and 2 patients with malignant 22-gauge needle aspirates as the sole identifier of paratracheal malignancy. As a staging tool, the 19-gauge needle was significantly more sensitive than the 22-gauge needle, 85.5 versus 52.7% (p = 0.0001). Overall, in 49 of 55 patients (89.1%) with malignant mediastinal lymphadenopathy paratracheal tumor was confirmed by TBNA. The 19-gauge TBNA staging of the mediastinum is an effective, safe, and cost-saving alternative to surgical mediastinal exploration that can be performed during initial diagnostic bronchoscopy.

Cold-dependent activation of complement: recognition, assessment, and mechanism.

Cold-dependent activation of complement (CDAC) is a phenomenon characterized by low hemolytic complement activity in chilled serum. Complement component levels are normal when measured immunologically, and there is normal hemolytic activity in EDTA plasma or serum maintained at 37 degrees C. Little attention has been paid to CDAC except in Japan, and current unfamiliarity with it, even by clinical immunologists, can lead to confusion and unnecessary laboratory tests. A 66-year-old patient with a complex medical history is described whose complement tests showed abnormalities characteristic of CDAC. Evidence for classical complement pathway activation in the cold was obtained by CH50 measurements, by hemolytic C4 determinations, by C4a, C3a, and C4d generation, and by quantitating C1s-C1r-(C1 inhibitor)2 complexes. A good correlation was observed among these parameters. Cryoprecipitates were absent. CDAC activity has persisted for over 5 years and is greater at 13 than at 4 degrees C. Activation is ablated by heating at 56 degrees C and restored by the addition of C1 to the heated serum. Adsorption by streptococcal protein G-Sepharose and precipitation by 2.5% polyethylene glycol support the hypothesis that CDAC is caused by aggregated IgG. The CDAC factor(s) also induces complement activation in normal serum but has not interfered with Raji cell or C1q binding tests or with FACS analysis. More limited studies of a second individual experiencing CDAC yielded similar results.

Interobserver variability in the interpretation of pulmonary artery catheter pressure tracings.

STUDY OBJECTIVE: We evaluated the ability of three independent reviewers (R1, R2, R3) using waveform analysis to accurately identify confirmed valid PCWP tracings, and their ability to consistently report the PCWP numerical value. DESIGN: Sixty PA and PCWP tracings were prospectively obtained and blindly reviewed by three independent critical care physicians. SETTING: The medical ICU of Wilford Hall USAF Medical Center. PATIENTS OR PARTICIPANTS: Twenty mechanically ventilated patients with PA catheters inserted for hemodynamic assessment. INTERVENTIONS: Sixty PA and PCWP tracings were reviewed blindly and independently for acceptability using waveform criteria by three critical care physicians. While recording all 60 tracings, blood was aspirated from the distal port of the PA catheter with the balloon "wedged" and blood gas analysis was done. Each reviewer analyzed the PCWP tracings for validity using waveform criteria, and reported a numerical PCWP reading for those tracings judged valid by waveform criteria. Reviewer sensitivity, specificity and accuracy in performing waveform analysis were assessed by comparing their predictions with those tracings that were confirmed their predictions with those tracings that were confirmed valid by the aspiration of pulmonary capillary blood. Inter-reviewer agreement upon which validity of PCWP tracings was based and reviewer agreement on the numerical PCWP reading were also assessed. All tracings were blindly reviewed by each physician, first without and then with an AP tracing to define end-expiration. MEASUREMENT AND RESULTS: Thirty-eight of 60 PCWP tracings were confirmed valid by the aspiration of pulmonary capillary blood. In the remaining 22 tracings, mixed venous blood was aspirated with the balloon wedged, and tracing validity was unconfirmed. Reviewer accuracy in identifying was 50 percent for R1, 65 percent for R2 and 57 percent for R3. No reviewer's accuracy was significantly different from a random guess which would yield an accuracy of 50 percent. Agreement by all three reviewers in identifying valid PCWP tracings using waveform analysis varied from 37 percent in the absence of an AP tracing to 66 percent when an AP tracing was available to identify end-expiration (p less than 0.003). Agreement by all three reviewers on the PCWP numerical reading (within 4 mm Hg) was 79 percent without an AP tracing and 96 percent with an AP tracing (p = NS). The numerical reading reported by the ICU nurses and house staff correlated closely with the reviewers' readings. Agreement with the reported PCWP reading was improved only for R2 by the addition of an AP tracing. CONCLUSION: We conclude that the validation of PCWP tracings by waveform analysis is subject to interobserver variability, and reviewer accuracy in identifying confirmed valid tracings was no better than a random guess. Agreement on the numerical PCWP reading was high among the reviewers as was agreement by each individual reviewer with the reported PCWP. Finally, the presence of an AP tracing, to define end-expiration, adds little to the interpretation of the PCWP numerical reading by experienced physicians.