Detecting pre-ovulatory luteinizing hormone surges in urine

The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre- ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.      

Tourniquet inflation after intra-articular morphine and bupivacaine administration following knee arthroscopy

We performed a randomized doubled-blind study to evaluate whether there was a benefit in delay in tourniquet deflation with intra-articular administration of morphine and bupivacaine following operative arthroscopic surgery. In 34 patients the tourniquet was deflated immediately and in 38 patients the tourniquet remained inflated for 10 min following injection. The analgesic efficacy was assessed using pain scores and the amount of supplementary analgesia required. The results demonstrate no benefit in delay in tourniquet deflation.     

Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity

Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.     

NADPH-dependent and -independent loss of cytochrome P-450 in control and phenobarbital-induced rat hepatic microsomes incubated with carbon tetrachloride

Carbon tetrachloride-mediated loss of cytochrome P-450 has been compared in hepatic microsomes from untreated and phenobarbital-treated rats. At concentrations of carbon tetrachloride greater than 2.5 mM, a direct effect (i.e., NADPH- independent) on cytochrome P-450 was observed. This apparently arose from the "solvent" properties of carbon tetrachloride as this effect could be duplicated with the physically similar alkyl halide 1,2-dibromo-3-chloropropane. NADPH-dependent loss of cytochrome P-450 occurred at lower concentrations with maximal response occurring at 2.5-5.0 mM. Residual cytochrome P-450 at these concentrations was similar in untreated and phenobarbital-treated microsomes. Mixed-function oxidase activities in phenobarbital-treated microsomes were reduced to levels below those of uninduced controls. The 52-kDa polypeptide(s) in untreated microsomes and that specifically induced in phenobarbital-treated microsomes were susceptible to NADPH-dependent carbon tetrachloride incubation. These data suggest that the susceptibility of specific forms of cytochrome P-450 to carbon tetrachloride can be duplicated in in vitro incubation. Furthermore, data on the direct action of carbon tetrachloride suggest that this route of damage must be taken into consideration when concentrations of carbon tetrachloride of 2.5 mM or greater are used in in vitro incubation systems.     

Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of alpha thalassaemia

AIMS: To assess the accuracy and precision of measuring haemoglobin A(2) by high performance liquid chromatography (HPLC) in the presence and absence of sickle cell trait, with or without alpha thalassaemia trait. METHODS: The haemoglobin A(2) percentage and the haemoglobin A(2) plus S percentages were determined by HPLC on 82 normal controls and 78 patients with sickle cell trait, respectively; the alpha thalassaemia status of each patient was determined by polymerase chain reaction. Red cell indices and haemoglobin A(2) and S percentages were compared in patients with two, three, or four alpha genes. RESULTS: Of the 78 patients with sickle cell trait, 17 were heterozygous for alpha(+) thalassaemia (-alpha(3.7)/alphaalpha) and 13 were homozygous (-alpha(3.7)/-alpha(3.7)). Microcolumn chromatography showed that the haemoglobin A(2) percentage was slightly, but significantly, higher than normal in sickle cell trait. HPLC determinations of haemoglobin A(2) percentage in patients with sickle cell trait are precise but inaccurate, the percentage being appreciably overestimated. The measured haemoglobin A(2) percentage is stable for one week, but inaccuracy increases by two weeks in most samples. Despite this inaccuracy, there are significant differences in the HPLC "haemoglobin A(2) percentage" between groups of individuals with two, three, and four alpha genes. CONCLUSIONS: Haemoglobin A(2) determinations by HPLC are precise but inaccurate. Nevertheless, there are significant differences in the haemoglobin A(2) percentage in subjects with two, three, and four alpha genes. Although there is some overlap between groups, this can be useful, together with the red cell indices, in predicting the likelihood of coexisting alpha thalassaemia.     

The presence of isoaspartic acid in beta-amyloid plaques indicates plaque age.

Extracellular deposits of fibrillar beta-amyloid are a characteristic neuropathology of Alzheimer's disease (AD). We have developed a novel antibody to a hypothesized "older isomer" of the amyloid protein. This antibody, raised against a synthetic beta-amyloid peptide containing isoaspartic acid at position 7 (isoaspartic-7-Abeta), reacts with isoaspartic-7-Abeta, a nonenzymatic modification found in long-lived proteins. Plaques stained with this antibody are thioflavine positive and are found throughout the frontal and entorhinal cortices of AD cases. In frontal cortex, isoaspartic-7-Abeta plaques are clustered but have a widespread distribution in all cortical layers. Isoaspartic-7-Abeta is found primarily in the core of individual plaques surrounded by nonisomerized amyloid. Activated microglia are associated with plaques containing isomerized and nonisomerized amyloid. In contrast to AD, isoaspartic-7-Abeta plaques in Down's syndrome (DS) cases are found primarily in the superficial layers of frontal cortex. Using image analysis isoaspartic-7-Abeta deposition was correlated with dementia severity in AD and with age in DS. The results indicate that this antibody against altered aspartyl amyloid could be a useful indicator of the age of amyloid plaques.