Partition of variation for predicting experimental power with a broiler chicken example.

A 1932 editorial in Poultry Science stated that sampling theory, or experimental power, could be useful for "the investigator to know how many … birds to put into each experimental pen." Nevertheless, in the past 90 yr, appropriate experimental power estimates have rarely been applied to research with poultry. To estimate the overall variation and appropriate use of resources with animals in pens, a nested analysis should be conducted. Bird-to-bird and separate pen-to-pen variances were separated for 2 datasets, one from Australia and one from North America. The implications of using variances for birds per pen and pens per treatments are detailed. With 5 pens per treatment, increasing birds per pen from 2 to 4 decreased the SD from 183 to 154, but increasing birds/pen from 100 to 200 only decreased the SD from 70 to 60. With 15 birds per treatment, increasing pens/treatment from 2 to 3 decreased SD from 140 to 126, but increasing pens/treatment from 11 to 12 only decreased the SD from 91 to 89. Choosing the number of birds to include in any study should be based on expectations from historical data and the amount of risk investigators are prepared to accept. Too little replication will not allow relatively small differences to be detected. On the other hand, too much replication is wasteful in terms of birds and resources, and violates the fundamental principles of the ethical use of animals in research. Two general conclusions can be made from this analysis. First, it is very difficult to detect 1% to 3% differences in broiler chicken body weight with only one experiment consistently because of inherent genetic variability. Second, increasing either birds per pen or pens per treatment decreased the SD in a diminishing returns fashion. The example presented here is body weight, of primary importance to production agriculture, but it is applicable whenever a nested design is used (multiple samples from the same bird or tissue, etc.).

In vitro and in vivo effects of lutein against cisplatin-induced ototoxicity.

INTRODUCTION: Cisplatin is a commonly prescribed drug that produces ototoxicity as a side effect. Lutein is a carotenoid with antioxidant and anti-inflammatory properties previously tested for eye, heart and skin diseases but not evaluated to date in ear diseases. AIM: To evaluate the protective effects of lutein on HEI-OC1 auditory cell line and in a Wistar rat model of cisplatin ototoxicity. MATERIALS AND METHODS: In vitro study: Culture HEI-OC1 cells were exposed to lutein (2.5-100 µM) and to 25 µM cisplatin for 24h. In vivo study: Twenty eight female Wistar rats were randomized into three groups. Group A (n=8) received intratympanic lutein (0.03 mL) (1mg/mL) in the right ear and saline solution in the left one to determine the toxicity of lutein. Group B (n=8) received also intraperitoneal cisplatin (10mg/kg) to test the efficacy of lutein against cisplatin ototoxicity. Group C (n=12) received intratympanic lutein (0.03 mL) (1mg/mL) to quantify lutein in cochlear fluids (30 min, 1h and 5 days after treatment). Hearing function was evaluated by means of Auditory Steady-State Responses before the procedure and 5 days after (groups A and B). Morphological changes were studied by confocal laser scanning microscopy. RESULTS: In vitro study: Lutein significantly reduced the cisplatin-induced cytotoxicity in the HEI-OC1 cells when they were pre-treated with lutein concentrations of 60 and 80 µM. In vivo study: Intratympanic lutein (1mg/mL) application showed no ototoxic effects. However it did not achieve protective effect against cisplatin-induced ototoxicity in Wistar rats. CONCLUSIONS: Although lutein has shown beneficial effects in other pathologies, the present study only obtained protection against cisplatin ototoxicity in culture cells, but not in the in vivo model. The large molecule size, the low dose administered, and restriction to diffusion in the inner ear could account for this negative result.

Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop.

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGß, hCGßn, hCGßcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGß-, 8 hCGα-, and 13 αß-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1%; epitope ß1), 12 with low (<1.0%; epitopes ß2/4), and 13 with high (>>1%; epitopes ß3/5) hLH cross-reactivity. The majority of hCGß epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of ß-sheet loops 1 and 3 (epitopes ß2-6; 27 mAbs) and the second around the cystine knot (e.g., epitopes ß1, ß7, and ß10; 9 mAbs). Four mAbs recognized epitopes on hCGßcf-only (e.g., epitopes ß11 and ß13) and six mAbs epitopes on the remote hCGß-carboxyl-terminal peptide (epitopes ß8 and ß9 corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGß-only, or hCG together with hCGß or hCG together with hCGß and hCGßcf. Sandwich assays that measure hCG plus hCGß and eventually hCGßcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGßcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope ß1 (Asp10, Asp60, and Gln89) in combination with epitopes ß2 or ß4 located at the top of ß-sheet loops 1 + 3 of hCGß involving aa hCGß20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.

A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases.

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.

Absorption of therapeutic drugs by barrier gels in serum separator blood collection tubes. Volume- and time-dependent reduction in total and free drug concentrations.

The stability of seven commonly monitored therapeutic drugs in serum was examined following storage in Vacutainer SST and Corvac serum separator blood collection tubes. Significant decreases (ranging from 5.9% to 64.5%) in the measured concentrations of phenytoin, phenobarbital, lidocaine, quinidine, and carbamazepine were observed, as a function of both time and sample volume, when serum was stored in Vacutainer SST serum separator blood collection tubes. In contrast, measured concentrations of theophylline and salicylate did not change under identical specimen storage conditions. No significant changes in the concentrations of phenytoin, phenobarbital, carbamazepine, theophylline, quinidine, and salicylate were observed when serum was stored in Corvac serum separator blood collection tubes. Only serum lidocaine concentrations decreased (ranging from 31.5% to 72.6%, depending on sample volume) after storage in Corvac tubes for 24 hours. The apparent decreases in serum concentrations of therapeutic drugs in both Vacutainer SST and Corvac tubes were most pronounced when small volumes (200-500 microL) of serum remained in contact with the barrier gels for prolonged periods of time (> 2-6 hours). These decreases were due to absorption of drugs by the barrier gels, as demonstrated by the recovery of drugs following chemical extraction of the barrier gels with methanol. For phenytoin and phenobarbital, the reduction in total drug concentrations also resulted in a proportional decrease in free drug concentrations and was dependent on the extent of protein binding by the drug. None of the therapeutic drugs used in this study were adversely affected by prolonged storage in standard red top Vacutainer blood collection tubes without barrier gels. The data suggest that serum separator blood collection tubes should be used with extreme caution for therapeutic drug monitoring, particularly when reduced sample volumes or prolonged specimen storage may be required.

Analytical performance evaluation of EMIT II monoclonal amphetamine/methamphetamine assay: more specificity than EMIT d.a.u. monoclonal amphetamine/methamphetamine assay.

We evaluated a new EMIT II monoclonal amphetamine/methamphetamine assay for screening human urine by comparing it with the EMIT d.a.u. monoclonal amphetamine/methamphetamine assay and a fluorescence polarization assay. The EMIT II assay has a cutoff of 1 mg/L d-methamphetamine. The EMIT II and EMIT d.a.u. assays were run on a BM/Hitachi 704 analyzer; for the fluorescence polarization assay we used a TDx analyzer. All EMIT II positive samples were also analyzed by the fluorescence polarization assay. We used gas chromatography/mass spectrometry (GC/MS) for confirmation of the presence of amphetamine or methamphetamine. Within-run CVs for the Level 1 (1 mg/L) and Level 2 (3 mg/L) calibrators for the EMIT II assay were 0.47% and 0.53%, respectively. Corresponding between-run CVs were 1.48% and 1.60%, respectively. Of the 1007 samples screened for amphetamines, 50 were positive by the EMIT d.a.u. assay; 21 samples (not a subset of the 50 samples) were positive by the EMIT II assay. However, 19 samples that tested positive by EMIT II also tested positive by the EMIT d.a.u. assay. Subsequent testing of the EMIT II positive samples by the fluorescence polarization assay detected in six positive samples. By means of chiral derivatization wer identified two specimens containing primarily l-isomers of amphetamine and methamphetamine. Sympathomimetic amines were identified in several of the samples not containing amphetamine or methamphetamine.

Analytical performance of EMIT cyclosporine assay evaluated.

We evaluated the EMIT Cyclosporine Assay (Syva Co., Palo Alto, CA), using the Cobas-Mira analyzer to assess the precision, accuracy, and analytical recovery from whole-blood samples supplemented with cyclosporine. We also performed comparative analysis of whole-blood samples containing cyclosporine from liver and kidney transplant patients by using EMIT, HPLC, and RIA (IncStar Cyclo-Trac, SP assay). Before assay by EMIT or RIA, cyclosporine was extracted from whole blood with methanol. For the HPLC method, whole blood containing cyclosporine was hemolyzed with 300 mL/L acetonitrile in water; cyclosporine was extracted from the hemolysate with acetonitrile. The within-run and between-run CVs for the EMIT assay of cyclospoprine were 9.9% (means = 72.6, SD = 7.2 micrograms/L; n = 20) and 13.5% (means = 75.0, SD = 10.1 micrograms/L; n = 26) for the low control; 3.5% (means = 194.7, SD = 6.8 micrograms/L; n = 20) and 8.1% (means = 189.0, SD = 15.3 micrograms/L; n = 26) for the medium control; and 7.0% (means = 332.5, SD = 23.3 micrograms/L; n = 20) and 7.1% (means = 340.0, SD = 24.2 micrograms/L; n = 24) for the high control (Bio-Rad, whole-blood controls). Analytical recovery of cyclosporine from drug-supplemented samples averaged 99% for EMIT, 104% for HPLC, and 90% for RIA over a concentration range of 50-500 micrograms/L. Analysis of 196 specimens by HPLC (x) vs EMIT (y) gave the following regression statistics: y = 1.27x + 16.44; IncStar's RIA (x') vs EMIT: y = 1.12x' - 2.50; HPLC vs RIA: x' = 1.10x + 23.87.

Monitoring free digoxin instead of total digoxin in patients with congestive heart failure and high concentrations of digoxin-like immunoreactive substances.

Digoxin-like immunoreactive substances (DLIS) are present in patients with conditions associated with volume expansion (including hypervolemic hypertension, renal failure, and liver failure) and in pre-eclampsia and premature birth. These strongly-protein-bound substances cross-react with anti-digoxin antibodies and cause falsely increased measured concentrations of digoxin in serum. Patients with congestive heart failure (CHF) often have volume expansion and are receiving digoxin therapy. They are also very sensitive to digoxin toxicity and have a very narrow therapeutic range (1.0-1.9 nmol/L). We found monitoring the concentrations of free digoxin (in protein-free ultrafiltrates) helpful in eliminating the interferences of DLIS in CHF patients. DLIS concentrations were measured by fluorescence polarization assay. Concentrations of DLIS were detectable in significantly more (58.3%) of the 12 CHF patients (group A) who were not receiving digoxin than in the 22 normal volunteers tested (13.6%) (P less than 0.05 by both chi-square and Fisher's exact test). Protein-free filtrates from patients or normal volunteers did not show any measurable DLIS activities. We also determined the concentrations of total and free digoxin in 12 patients with CHF who were receiving digoxin (group B) and compared the results with those for 22 patients receiving digoxin without the diagnosis of CHF or any known pathological conditions that could increase DLIS concentrations. The ratio of free to total digoxin in patients in group B was significantly lower (mean = 52.8%, SD 10.2%) than in those receiving digoxin (mean = 72.7%, SD 6.5%) for other reasons (independent two-tailed t-test, P less than 0.05).

Elevated lipid peroxidation products and depleted transferrin levels in the plasma of kidney transplant recipients.

Lipid peroxidation products were measured in the plasma of 24 kidney transplant patients and 12 healthy volunteers (controls) by: (1) 2-thiobarbituric acid assay and (2) the intensity of fluorescence products of malonaldehyde cross-linked proteins. Plasma levels of creatinine, ceruloplasmin, transferrin, prealbumin, albumin and total protein were also measured. Elevated lipid peroxidation products and lowered transferrin levels were observed in transplant patients compared to controls. Ceruloplasmin levels were slightly but significantly elevated in recent transplant recipients (less than 6 months, n = 12, Group A) while no difference was observed between older transplant recipients (greater than 6 months, n = 12, Group B) and controls. Serum, creatinine levels were also slightly but significantly elevated in both groups of patients compared to controls. Serum prealbumin, albumin and total protein levels in both groups of transplant recipients were not different from controls or reference range values.

[Evaluation of antacids "in vitro"]. / Evaluación de antiácidos "in vitro".

Five different antacids were compared "in vitro" through titration with NaOH 0.5 N to observe their neutralising power towards HCl 1.0 N. Eight pH measurements were done for each antacid. The neutralising capacity of the antacid was calculated with a specific formula for each pH measure reading, with this information statistical calculation were made to compare the antacids among themselves, with suggest that the method can be useful in the valuation of newly produced antacid before their clinical application.