Use of alkaline phosphatase to correct the underestimation of fosphenytoin concentration in serum measured by phenytoin immunoassays

The pharmacologic activity of fosphenytoin, a new phosphate ester pro-drug of phenytoin, is due to in vivo conversion to phenytoin. Fosphenytoin concentrations cannot be accurately estimated by phenytoin immunoassays (fluorescence polarization and chemiluminescence) owing to the nonlinear relation between fosphenytoin concentration and the observed cross-reactivity. The problem of slow conversion of fosphenytoin to phenytoin in serum in vitro can be circumvented by rapidly converting fosphenytoin to phenytoin in vitro by alkaline phosphatase. Drug-free serum, heparin, EDTA, or citrated plasma were supplemented with 2 concentrations of fosphenytoin. Then to 1-mL aliquots of specimen, no enzyme (control), 10 microL, or 25 microL of enzyme solution was added. The specimens were incubated, and phenytoin concentrations were measured by fluorescence polarization and chemiluminescent assays. In the absence of enzyme, we observed little conversion of fosphenytoin to phenytoin, but in the presence of only 10 microL of enzyme, the conversion of fosphenytoin to phenytoin was complete in 5 minutes. We also observed complete conversion of fosphenytoin to phenytoin by alkaline phosphatase in heparin, EDTA, and citrated plasma. If clinically indicated, the phenytoin concentration can be measured before and after addition of enzyme to roughly estimate the rate of conversion.     

Stimulus-dependent leukotriene release from human basophils: a comparative study of C5a and Fmet-leu-phe

Previous studies of human basophil mediator release have noted that the bacterial peptide fmet-leu-phe and the anaphylatoxin C5a induce comparable levels of histamine release while only fmet peptide induces leukotriene release. Since 5-lipoxygenase metabolism of arachidonic acid is calcium dependent, we examined the characteristics of the human basophil [Ca++]i response which follows its activation by either fmet peptide or C5a. While the peak [Ca++]i response was essentially identical for these two stimuli, fmet peptide induced a prolonged increase in [Ca++]i while C5a stimulated only a transient increase in [Ca++]i that was essentially over within 2 minutes of adding the stimulus. Simultaneous addition of EDTA with fmet peptide revealed the two phases of the [Ca++]i response and demonstrated that leukotriene release was dependent on an elevated [Ca++]i level in the 2-5 minutes following challenge. Enhancement of leukotriene release induced by C5a by agents such as staurosporine and interleukin-3 also produced a [Ca++]i kinetic curve which resembled fmet peptide. Single cell studies of the [Ca++]i response could detect no subpopulations of cells which responded preferentially to fmet peptide or C5a, eliminating the possibility that the ability of fmet peptide to induce leukotriene was a result of its action on a functionally distinct population of basophils.     

Action of some foreign cations and anions on the chloride permeability of frog muscle

1. Evidence for the existence in skeletal muscle of a specific cation binding system capable of lowering the chloride permeability was obtained by testing the effect of several metal ion species upon the efflux of (36)Cl from frog muscles equilibrated in high-KCl solution.2. Cu(2+), Zn(2+) and UO(2) (2+) ions, when present in concentrations of approximately 10(-4)M in inactive wash solution at pH 7.4 slowed the efflux of (36)Cl to half its original value. At pH 5.0, when the chloride permeability was already low as a consequence of hydrogen ion binding, these metal ions had little further effect.3. Presence of Ni(2+), Co(2+), Pb(2+), Ce(3+) and La(3+) in 10(-4)M or higher concentrations had no detectable influence on the (36)Cl efflux. Wide variations in Ca(2+) concentration were similarly ineffective.4. The influence of more adsorbable anions on the chloride permeability was examined at different pH values. Extracellular iodide greatly slowed the rapid efflux of (36)Cl into alkaline solution. In acid solutions, when the chloride permeability was already low, the effect of iodide was less pronounced, but still demonstrable. The chloride permeability was consequently increased to a lesser extent by a rise in pH in the presence of iodide.5. The efflux of iodide and bromide was measured at different pH values under conditions of self exchange. In alkaline solution the permeabilities to iodide and bromide were considerably lower than that to chloride. In acid solution the membrane differentiated less between anion species of different adsorbability.     

Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency

An infant girl with elevated blood lactate, pyruvate, and plasma branched-chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3; dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) deficiency. Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cultured skin fibroblasts, respectively. Western blot analysis demonstrated that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis showed normal amounts of E3 RNA. DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles. One is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutation which results in the premature termination of the precursor E3 polypeptide (Y35X). The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activity probably by structural change in the E3 dimer. DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was inherited from the father and the missense mutation was inherited from the mother.      

In vivo administration of anti-CD4 monoclonal antibody prolongs survival in longtailed macaques with experimental allergic encephalomyelitis

The in vivo administration of monoclonal antibody (mAb) to the CD4 antigen associated with helper T cells has been successful in prolonging the survival of nonhuman primates with experimental allergic encephalomyelitis (EAE). EAE was induced in 17 outbred longtailed macaques (Macaca fascicularis) by inoculation of homologous myelin basic protein (BP) in complete Freund's adjuvant (CFA). Treatment was begun at the onset of clinical signs. Eleven animals were treated with anti-CD4 mAb Leu3a (eight) or OKT4a (three). Of the six control animals, two received anti-CD8 mAb (Leu2a), and four were treated with saline. Specific T- and B-cell subsets which have been implicated in the development of EAE were monitored throughout the course of the disease by one- and two-color immunofluorescence (IF). The monkey anti-BP antibody and anti-mouse immunoglobulin (IgG) responses were measured by enzyme-linked immunoassay (ELISA) techniques, as were the levels of free-circulating murine IgG. The nature of the infiltrating lymphocytes in the brain was evaluated histologically post mortem. Our results indicate that anti-CD4 mAb can prolong survival and in some cases completely reverse the clinical appearance of the disease; however, relapses did occur. Treatments with Leu3a or OKT4a anti-CD4 mAbs reversed the ongoing depletion of CD4+ and CD8+ cells caused by the development of EAE and appeared to reduce the size and degree of inflammation in brain lesions. These treatments did not induce immunologic tolerance to mouse IgG since all of the anti-CD4-treated animals produced high titers of anti-mouse IgG antibodies. Treatment with Leu2a (anti-CD8) had no effect on the development of EAE. These results suggest that CD4+ cells are important to the pathogenesis of EAE in macaques and that manipulation of this subset with monoclonal antibodies may provide effective treatment of human demyelinating disease.     

Patients'' complaints and satisfaction 5 years after complete denture treatment

This study is based on an evaluation of patients' opinions of their dentures by means of a questionnaire. The patients had been provided with complete dentures at a university clinic. Of the original sample (n = 139) a test group of 92 patients remained available for the actual study 5 yr after treatment. The replies in the questionnaire were subjected to statistical processing and interpretation. Six clusters could be defined and these were introduced as "scales" representing the patients' score on a specific type of complaint. Apart from the six scales a score was achieved for each patient's overall satisfaction. The correlations between the various scales were calculated with the aid of a computer program. Finally, cross-tabulation was used to see if any significant (inter)correlations could be proved between complaints, satisfaction, and some of the patients' social data. It could be concluded that a correlation exists between complaints of pain and of functional aspects on the one hand and vague complaints on the other hand. There is also a correlation between complaints of looseness of the maxillary denture and complaints about aesthetic factors. Well-fitting and well-functioning dentures, absence of pain, and a socially acceptable appearance contribute much to the patients' satisfaction with dentures. Social variables such as type of insurance (public or private) and marital status influence denture appreciation to a certain extent.     

Research review: a computer-based diagnostic model for individual case review

A previous study found that Iliad, a diagnostic expert system, detects diagnostic errors missed by peer review organization (PRO) review. That study used volunteer physicians from an institution as gold standard reviewers, however. The article discusses a second experiment employing Utah PRO (UPRO) review physicians as gold standards. Iliad was compared with the Unified Clinical Data Set used by the UPRO and was found to detect otherwise unsuspected diagnostic errors. The confirmation rate of Iliad flags was much higher in the earlier study, however. No agreement was found between institution and UPRO physicians, but there was agreement between a unique physician (who was both an institution and UPRO physician) and each of the two groups. Because Iliad screens for potential diagnostic errors to be confirmed or denied by gold standard physician review, the different types of physicians in the two experiments might have been the cause.