An enzyme-linked immunosorbent assay for the quantitative determination of anti-D in plasma samples and immunoglobulin preparations.

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40-5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.

Predictors of response to alprazolam and placebo in patients with panic disorder.

Date from a panic disorder treatment study with 506 patients, comparing alprazolam and placebo in a double-blind manner for 8 weeks, were analyzed to identify demographic and clinical characteristics of the patients that might predict response to treatment. The strongest predictors of response to alprazolam were age over 40, and lower baseline levels of anxiety and phobic symptoms. Predictors of response to placebo were weaker and, in addition to lower levels anxiety and panic attacks, included a lack of previous psychiatric treatment.

[Embolism of the aortic bifurcation. Complication of bacterial endocarditis in childhood]. / Aortale Sattelembolie. Komplikation einer bakteriellen Endokarditis im Kindesalter.

Acute occlusion of the aortic bifurcation by a sattle embolus, as a complication of infective Endocarditis, is a very rare, extremely serious event. The treatment of choice consist in an immediate simultaneous bilateral femoral embolectomy. The indication for operation must be realised without delay based on anamnesis and physical examination.

Calcemic status of geriatric patients: a longitudinal study.

Serum ionized (CaI), total (CaT) and albumin-'corrected' total (CaA) calcium concentrations were measured in 108 geriatric long-term inpatients on admission and again 37 months (mean) later. Mean CaI, especially when pH adjusted, remained extremely stable, indicating well-preserved homeostatic calcemic control even in the terminal years in the elderly. Further, the results indicate that in addition to their previously shown invalidity in interindividual comparisons, CaT and CaA are inferior to CaI and can also be misleading in the detection of slight longitudinal changes in the 'true' calcemic status of an individual geriatric patient.

Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva.

We have developed specific immunoassays for secretory peroxidase (SP) and for myeloperoxidase (MP) (polymorphonuclear leukocyte-derived peroxidase) in human saliva. Antibodies against SP and MP were produced using bovine milk lactoperoxidase (LP) and human MP as the immunogens, respectively. The methods developed are non-isotopic immunometric assays using biotinylated antibodies and avidin-enzyme conjugate. The detection limit was 0.1 ng/ml and the performance time less than 3 h for both assays. The determination ranges were 0.5-100 ng LP (SP)/ml and 0.5-200 ng MP/ml with intra- and interassay CVs of 4.3% and 15.6% for SP and 3.7% and 10.8% for MP, respectively. The mean analytical recoveries were 108.9% (SP) and 91.5% (MP). These assays correlated well (r = 0.849-0.871) with the colorimetric assays based on the oxidation of thiocyanate or chloride by peroxidases. However, compared to the colorimetric methods the new immunometric assays are much more sensitive and specific for salivary SP and MP. The assays are also more rapid since extensive dialysis to remove endogenous thiocyanate is not required.

Crevicular fluid myeloperoxidase--an indicator of acute gingival inflammation.

Paper strips inserted into 100 gingival sulci or periodontal pockets in 9 patients for 3 minutes in each case were assayed for polymorphonuclear (PMN) leukocyte-derived myeloperoxidase (MP) activity. This activity was then analysed in relation to the simultaneously recorded gingival index, probing depth and bleeding on probing. MP activity was always very low if the gingiva was clinically healthy, with no bleeding or signs of loss of attachment. However, sites at which the marginal gingiva was inflamed and bleeding and/or the pocket depth was greater than or equal to 6 mm exhibited MP activities ranging from very low to very high. The wide variation most likely reflects differing numbers of PMN cells in these crevices and is, therefore, probably indicative of acute or chronic periodontal disease at any particular site. The assays of the paper strips resulted in values linear with respect to the actual concentrations of purified, PMN cell-derived MP in the strips. Our method is a relatively simple way to quantitate the products of PMN cells at a single periodontal site and, in this way, to possibly identify the acute phases of the disease.

Optical and EPR spectroscopy studies on haem arginate, a new compound used for treatment of porphyria.

A protohaem compound, used for treatment of porphyrias, has been studied to elucidate its state of aggregation. EPR and absorption spectroscopy measurements reveal that 38.3 mM protohaem, dissolved in 40% 1,2-propanediol/10% ethanol/water solution, also containing 153 mM arginine, is partly EPR silent. It exists as high molecular weight aggregates and probably also as mu-oxo-dimers. Dilution in the aqueous alcohol solution dissolves the aggregates first to oligomers and dimers, and finally to monomers (Kdiss = 24 X 10(-6)M). When haem is diluted in 0.9% sodium chloride, a fully monomeric state is not reached even at 1 microM concentration. At 3.5 microM concentration, that used for infusion in patients, the haem is still totally aggregated.

Antibacterial effect of lactoperoxidase and myeloperoxidase against Bacillus cereus.

An oral periodontopathic bacterium, Bacillus cereus, was inhibited both by lactoperoxidase (LP) and myeloperoxidase (MP) antimicrobial systems. With the LP-SCN--H2O2 system, the growth inhibition was directly proportional to the amount of OSCN- ions present. The OSCN-, which is the principal oxidation product of the LP (or MP)-SCN--H2O2 system at neutral pH, is a normal component of human saliva. The oxidation products of both peroxidase systems inhibited the growth of the bacteria. This inhibition was associated with reduced extracellular release of collagenase activity from the cells. With LP, the antimicrobial efficiency of the oxidizable substrates was SCN- greater than I-, and with MP, the efficiency was I- greater than Cl- greater than SCN-, respectively. LP did not oxidize Cl-.

Isolated atrioventricular discordance. Report of two surgical cases with isolated ventricular inversion.

Isolated ventricular inversion is a term used for a congenital heart malformation with the segmental arrangement atrioventricular (AV) discordance and ventriculoarterial concordance. It describes a condition which from a physiological point of view resembles complete transposition of the great arteries. We have recently seen two patients with this anomaly. Both underwent intracardiac repair by means of a Mustard operation at 10 years and 10 months of age, respectively. Associated lesions in the first patient were an AV septal defect with two AV orifices (partial AV canal, ostium primum defect), partial anomalous pulmonary venous return, and anomalies in the systemic venous drainage. A perimembranous ventricular septal defect complicated the condition in the second patient.

A comparative study of the low-temperature magnetic circular dichroism spectra of horse heart metmyoglobin and bovine liver catalase derivatives.

The magnetic circular dichroism (MCD) spectra of three horse heart metmyoglobin compounds, the cyanide, azide and hydroxide forms, have been measured in the visible and near infrared spectral regions at temperatures down to 1.5 K. The three compounds are all virtually completely low-spin at low temperatures with ground g factors of decreasing rhombicity in the order CN- greater than N3- greater than OH-. The MCD magnetization curves have been constructed at selected wavelengths throughout the visible and near infrared regions. The curves are independent of wavelength, showing that all the bands studied are x,y polarized and can, moreover, be satisfactorily fitted to the g factors determined by EPR spectroscopy with theoretical expressions (Thomson, A.J. and Johnson, M.K. (1980) Biochem. J. 191, 411-420). This confirms the assignment and polarizations of the near infrared region low-spin ferric haem charge-transfer bands. The energies of these transitions are markedly dependent upon the added axial ligand, ranging from 1595 to 1295, and 1050 nm for the compounds CN-, N3- and OH-. The MCD spectra of bovine liver catalase and its cyanide adduct have been recorded in the Soret, visible and near infrared regions. Catalase is know to have phenolate anion as the proximal ligand of the haem group. The forms of the spectra make an interesting comparison with those of the analogous metmyoglobin derivatives, in which histidine is the proximal ligand. The MCD spectra of catalase at 4.2 K is an example of a fully high-spin haemoprotein. The cyanide compound is completely low-spin at 4.2 K. The near infrared charge-transfer band is at 1300 nm, showing the effect on the energy of this band of changing from imidazole to phenolate ion as the proximal ligand to haem.

Assignment of the axial ligands of the haem in milk lactoperoxidase using magnetic circular dichroism spectroscopy.

The absorption and MCD spectra of ferric lactoperoxidase from milk and its cyanide and fluoride derivatives have been measured in the near infrared and visible wavelength regions both at room temperature and at 4.2 K. By comparison with the MCD spectra of haemoproteins of known axial ligation, which also contain protohaem IX, it has been possible to arrive at suggestions for the axial ligation in lactoperoxidase. At room temperature oxidized lactoperoxidase has the haem iron in the high-spin state, and the results indicate that the proximal ligand of the haem iron is a histidine imidazole and that the sixth ligand is probably a carboxylate ion. At 4.2 K oxidized lactoperoxidase converts almost totally to a low-spin form, changing the sixth ligand to a histidine imidazole, which is in the imidazolate form.

The heme environment of leghemoglobins. Absorption and circular dichroism spectra of artificial leghemoglobins and myoglobins.

Artificial leghemoglobins were reconstituted from apoleghemoglobin and meso-, deutero- and diacetyldeuteroheme. Absorption and circular dichroism spectra of their high-spin and low-spin derivatives in the ferrous and ferric forms were recorded in the ultraviolet and visible wavelength regions. The substitution of the 2,4-side-chains of heme induced changes in the optical activity, reflecting alterations in the heme environment. The effect on the conformation of aromatic amino acid residues around heme obviously correlates with the sixth axial ligand and the spin state of iron. Absorption and CD spectra of the aquoferric derivatives of artificial myoglobins were recorded in comparison. Strongly electron-withdrawing acetyl side-chains at the 2,4-positions of diacetyldeuteroheme caused a change in the absorption spectra of aquoferric leghemoglobin and myoglobin towards low spin. On the basis of the spectra it was suggested that the displacement of the ferric iron from the pyrrole plane in leghemoglobin derivatives would be smaller than in the corresponding myoglobin derivatives.

Structure of milk lactoperoxidase. A study using circular dichroism and difference absorption spectroscopy.

Circular dichroism spectra of milk lactoperoxidase, its fluoride and cyanide derivatives, and those of ferrous lactoperoxidase and its carbonyl and cyanide compounds, were recorded in the wavelength region 200-670 nm. All derivatives have split ellipticity bands, suggesting that lactoperoxidase has a narrow heme pocket that prevents ligands forming linear iron-ligand bonds. Difference absorption spectroscopy of the enzyme in the far-ultraviolet region supports the previously held view that the fifth ligand of the heme iron is histidine. The secondary structure of lactoperoxidase, calculated from the far-ultraviolet circular dichroism spectrum, contains 65% beta-structure, 23% alpha-helix and 12% unordered structure. Reduction of lactoperoxidase with dithionite gives two forms, indicating that after reduction some compound arising from dithionite binds in the vicinity of the heme iron.

Resonance Raman study on yeast cytochrome c peroxidase. Effect of coordination and axial ligands.

Resonance Raman spectra are reported for native ferric cytochrome c peroxidase, its cyanide and fluoride compounds, those of the ferrous enzyme and its cyanide and carbonyl compounds, and the spectrum of the hydrogen peroxide compound, compound I. Band frequencies of ferric horseradish peroxidase isoenzyme C2 and its derivatives are also given. Comparison of the frequencies of the bands around 1400, 1500, 1560-1580, and 1610-1640 cm-1 with those of other hemoproteins and heme model compounds showed that in ferric highspin compounds in particular the bands are not only spin and oxidation sensitive, as has previously been reported, but that they also reflect the coordination of the heme iron. It is suggested that ferric cytochrome c peroxidase and horseradish peroxidase are pentacoordinated. In the hexacoordinated fluoride, cyanide and carbon monoxide derivatives the bands reflect the spin state and the out-of-plane position of the heme iron. The spectrum of cytochrome c peroxidase compound I supports previous studies that suggest that it has a lowspin heme iron in the Fe(IV) oxidation state.