Novel calibration of a dynamic surface tension detector: flow injection analysis of kinetically-hindered surface active analytes.

First, a novel technique for calibration of a dynamic surface tension detector (DSTD) is described. The DSTD measures the differential pressure as a function of time across the liquid-air interface of growing drops that repeatedly form and detach at the end of a capillary tip. The calibration technique utilizes the ratio of pressure signals acquired from the drop growth of two separate solutions, i.e. a standard solution and a corresponding mobile phase, such as water, both of which have a known surface tension. Once calibrated, the dynamic surface tension of an analyte is obtained from the ratio of the pressure signals from the analyte solution to that of the mobile phase solution. Thus, this calibration technique eliminates the need to optically image the radius of the expanding drop of liquid. Accurate dynamic surface tension determinations were achieved for aqueous sodium dodecyl sulfate (SDS) solutions over a concentration range of 0.5-5.4 mM. The measured surface tensions for these SDS solutions range from 70.3 to 46.8 dyne/cm and were in excellent agreement with the literature. A precision of 0.2 dyne/cm (1 S.D.) was routinely obtained. Second, the DSTD with this calibration technique was combined with flow injection analysis (FIA) for the study of model protein solutions and polymer solutions. The kinetic surface tension behavior of aqueous bovine serum albumin (BSA) solutions as a function of concentration and flow rate is presented. Evaluation of the dynamic surface tension data illustrates that a protein such as BSA initially exhibits kinetically-hindered surface tension lowering, i.e. a time dependence, as BSA interacts with the liquid-air interface of an expanding drop. FIA/DSTD is then shown to be an effective tool for the rapid study of kinetically-hindered surfactant mixtures. It was found that mixtures of SDS and the polymeric surfactant Brij(R)-35 (lauryl polyoxyethylene ether with an average molecular weight of 1200 g/mol) result in essentially an additive lowering of the surface tension. Mixtures of polyethylene glycol (PEG), with an average molecular weight of 1470 g/mol, and Brij(R)-35, however, result in a competitive (non-additive) surface tension with the Brij(R)-35 dominating the response.

Screening for alpha-thalassemia. Correlation of hemoglobin H inclusion bodies with DNA-determined genotype.

We evaluated potential screening protocols for alpha-thalassemia in a group of 80 patients whose genotypes were determined by Southern blot analysis with alpha- and zeta-globin DNA probes. Erythrocyte inclusion bodies were measured by a modified brilliant cresyl blue test. Erythrocyte indices and iron status were also measured. The brilliant cresyl blue test reliably detects couples at risk for hemoglobin Bart's hydrops fetalis. Measurement of the number of inclusion bodies differentiates the alpha-thalassemia genotypes in the absence of a coincident beta-chain synthesis deficiency, such as hemoglobin E or beta-thalassemia. The test appears to identify patients, such as those with the Thai and Filipino deletion variants, whose alpha-thalassemia cannot be definitively characterized by DNA testing when only alpha- and zeta-globin probes are used in the analysis. We also found evidence of elevated serum ferritin levels in many patients with deletion of two or three alpha-globin genes. This study shows that most routine screening for alpha-thalassemia can be performed with three simple tests: (1) the brilliant cresyl blue inclusion study, (2) erythrocyte indices, and (3) iron studies. Analysis with DNA probes is needed in only some circumstances.

Death following accidental sodium azide ingestion.

Two college students developed symptoms of poisoning following ingestion of a salt solution during a college physiology laboratory exercise. Symptoms included nausea, vomiting, diarrhea, and altered consciousness. The ingested solution was identified as isotonic buffered saline containing sodium azide in a concentration of 1.0 g/L. The solution was commercially prepared for instrumentation use only and was used inadvertently for the exercise instead of freshly preparing sodium chloride in water. One student drank three sips of the solution and survived. The other student drank 700 to 800 mL and over several days became progressively ill, suffering myocardial damage and cardiac dysrhythmias, and, finally, died. Toxicologic studies confirmed the presence of azide in an antemortem urine sample from the deceased. Sodium azide is an uncommon but potent poison which can cause serious illness and death.

Development and evaluation of a simplified dot-blot method for detecting the delta F508 mutation in cystic fibrosis.

The recent discovery of the cystic fibrosis gene enables DNA-based testing for the direct identification of the deletion of three basepairs coding for phenyalanine at codon 508, the major mutation responsible for the disease. This mutation can be detected by analysis of amplified DNA with allele-specific oligonucleotide probes. We have simplified the procedure of Kerem et al. (Science 1989;245:1073-80) so that the assay can be routinely completed in one working day, starting with an extracted DNA sample. Addition of salmon-sperm DNA to the product of the polymerase chain reaction greatly improved the quality of the hybridization signal. The precision of the method was evaluated by blind analysis and interpretation of results for 100 specimens from 25 patients. The same result was obtained for each patient analyzed separately four times, and four independent observers agreed on the interpretation of results for all 100 specimens. No specimen required repeat analysis to produce interpretable results. We conclude that this method is reliable and convenient for routine clinical laboratory use.

Laser photobioactivation mechanisms: in vitro studies using ascorbic acid uptake and hydroxyproline formation as biochemical markers of irradiation response.

Clinical investigations of laser photobioactivation, or biostimulation, might be differently designed and more fruitful if knowledge of basic biochemical mechanisms were better understood. In this investigation, biochemical events identified as responses to 904 nm irradiation included increased ascorbic acid uptake by fibroblasts. These cells also showed increased hydroxyproline formation, and this was increased several-fold by the addition of proline to the medium. Maximum biochemical responses were observed at a pulse frequency of 67 Hz and a pulse width of 150 nsec with an energy density of approximately 7 mJ/cm2 per exposure. Elements in the mitochondrial cytochrome system are proposed as the radiation absorbing chromophore(s). Hypothetically, the energy generated is linked to ascorbic acid uptake, which in turn stimulates collagen synthesis.

Genetic screening of newborns for sickle cell disease: correlation of DNA analysis with hemoglobin electrophoresis.

Although DNA analysis based on the polymerase chain reaction (PCR) offers potential advantages for screening newborns for sickle cell disease, few data are available concerning the reliability of PCR-based tests for such screening. We describe a protocol for detecting the A, S, and C alleles of the beta-globin gene in dried blood from phenylketonuria screening cards. This method is based on PCR and detection with allele-specific oligonucleotide probes. Results of a blind comparison of PCR analysis of the dried blood with hemoglobin electrophoresis of whole-blood samples agreed for 80 of 81 samples. The single discrepancy is probably not attributable to a failure of the PCR method, but rather to limitations of the electrophoresis method. The PCR method should be a highly accurate means of detecting beta-globin alleles in routine genetic screening with dried blood already collected for (e.g.) phenylketonuria screening.

Dynamic surface tension and adhesion detection for the rapid analysis of surfactants in flowing aqueous liquids.

A multidimensional dynamic surface tension detector (DSTD) for flowing liquid samples is reported. The DSTD is based on measuring the pressure with time of a repeating drop formed by the flow of liquid out the end of a pointed stainless steel capillary. This pressure-based DSTD provides information on dynamic surface tension at the air-liquid interface and adhesion at the solid-liquid interface on the side of the pointed capillary tip for each drop of surfactant solution, resulting in rapid characterization of complex samples. The signal obtained with the pressure-based DSTD is characterized, and a method is developed for extraction of the desired analytical information from the pressure signal. The DSTD was calibrated with 2-propanol over the range of 0-0.30 in relative surface tension lowering, Δγ/γ. The experimentally obtained Δγ/γ was in agreement with a theoretical model and published data for Δγ/γ over this range. A data analysis method was developed to adapt the DSTD to applications such as liquid chromatography and flow injection analysis, where the concentration of surfactant changes as a function of time. The DSTD signal yields a pressure-based Δγ/γ that is due to surface tension alone and a time-based Δγ/γ that is a combination of both surface tension and adhesion, providing essentially a contact angle measurement on a flowing sample. The data analysis method involves plotting the pressure-based and time-based surface tension measurements against each other at the same surfactant concentration for each pair of measurements, yet over a range of concentrations to establish a slope. This is referred to as a dynamic analysis plot and is applied in the characterization of various surfactants such as dodecyl sulfate ion-paired with tetrabutylammonium, industrial surfactant solutions FC-171 and FC-129, and biological surfactants tetradecyl maltoside, benzyldimethyldodecylammonium bromide, and 3-(N-decyl-N,N-dimethylammonio)-1-propanesulfonate. The slopes of the dynamic analysis plots for these surfactants were found to be unique, generally independent of concentration, and useful for understanding the type and degree of operating surface interactions. The FC-171 solution was found to exhibit considerable adhesion at the capillary tip, while dodecyl sulfate was found to have a small adhesion effect. Adhesion for dodecyl sulfate solutions was significantly enhanced by coating the stainless steel capillary tip with a hydrophobic polymer. Thus, there is potential for tuning the extent of the surface tension and adhesion effects for selective chemical analysis. The detection limit for dodecyl sulfate ion-paired with tetrabutylammonium is 0.9 ppm. Application of the DSTD for liquid chromatography is demonstrated, and the multidimensional data are shown to be useful in identifying and characterizing the poly(ethylene glycol)s separated from 2-propanol.

Chemiluminescent measurement of total urinary nitrogen for accurate calculation of nitrogen balance.

This instrumental method for total urinary nitrogen (TUN) is based on the principle of gas-phase chemiluminescence. Results correlate well with measurements of TUN by the Kjeldahl method, which has long provided the means to calculate nitrogen balances for nutritional management. In recent years, because of speed and convenience of measurement, determination of urinary urea nitrogen (UUN) has been substituted for Kjeldahl TUN. However, in patients requiring aggressive nutritional support, the UUN may not be a valid indicator of total nitrogen excretion. We compared nitrogen balances calculated for patients, using both UUN and chemiluminescence TUN data. For both normal and hospitalized populations, nitrogen balance calculated from UUN data exceeded that calculated from TUN data. We show that use of UUN data in calculating nitrogen balance may result in an incorrect assessment of many patients as being in positive nitrogen balance. TUN determined by chemiluminescence evidently provides a simple means of calculating nitrogen balance more nearly accurately.