The in vitro serum protein-binding characteristics of bis-(2-ethylhexyl) phthalate and its principal metabolite, mono-(2-ethylhexyl) phthalate.

The metabolism and toxicity of the ubiquitous plasticizer, bis-(2-ethylhexyl) phthalate (DEHP), and its principal metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been extensively investigated. In an attempt to understand their disposition in man, we studied the in vitro serum protein-binding characteristics of these compounds, using ultracentrifugation and agarose gel electrophoresis. The association of DEHP and lipoproteins was shown to be highly dependent upon, and proportional to, the lipid concentration of the serum. It appears that more than half of the serum DEHP is bound to proteins with density greater than 1.21 g/mL when the concentration of cholesterol is below 300 mg/dL or the cholesterol and triglyceride total concentration is less than 600 mg/dL. As the cholesterol and triglyceride concentrations increase, the percent DEHP bound to VLDL, IDL, and LDL increases. MEHP is bound principally to nonlipoprotein constituents in the serum, and this binding distribution is unaffected by lipid concentration. The percent binding of DEHP and MEHP to individual proteins was also found to be unaffected by their concentrations in serum. These data indicate that the protein-binding characteristics of these compounds, in vitro, is somewhat more complex than previously reported.

The performance of protein, lactic dehydrogenase, and alkaline phosphatase electrophoresis as part of a hospital admission screening procedure.

The practice of pre-admission hospital laboratory testing has been criticized as inefficient and cost ineffective. Laboratory screening is frequently condemned without regard for the possibility that a particular screening protocol may be poorly conceived and inadequate. To increase the specificity of our screening procedure to answer some questions raised by results of total enzyme and protein results, we have adapted the Nerenberg "sandwich" technique for multi-sample, simultaneous electrophoresis of sera for proteins, lactic dehydrogenase, and alkaline phosphatase. Twenty-six samples can be analyzed per hour by one technician at a total cost of about $1.00 per specimen. Results are interpreted visually without densitometry. Addition of the technique to our laboratory screening procedure has produced a demonstrable increase in test specificity and sensitivity. There is also a potential for a significant reduction in laboratory costs through anticipation and reduction of individually-performed electrophoretic techniques.

The albumin binding of unconjugated bilirubin in serum.

1. The limited bilirubin binding capacity of human serum albumin, and the fact that kernicterus can occur once the serum unconjugated bilirubin concentration exceeds this capacity, makes the assessment of non-albumin bound free bilirubin valuable in cases of severe neonatal hyperbilirubinemia. 2. Present methodology for this assessment utilizes Sephadex column chromatography, and is somewhat tedious and slow. 3. We have developed a procedure for assessing the albumin binding capacity of serum by titrating a sample of the serum with T-20 Dextran coated charcoal. 4. The method requires 2 ml of serum, takes 90 minutes to complete and is highly reproducible. 5. By this method, we can determine the reported secondary loose binding capacity of the albumin as well as the tight binding capacity which is determined by existing methods. 6. The tight binding capacity of a pool of normal adult human serum was found to be 20 mg/dl of serum. 7. This is in agreement with existing methods. The loose binding capacity was found to be an additional 10 mg/dl of serum. Added phenobarbital was found to lower the tight binding capacity, but not the secondary capacity.

Simultaneous assay of diazepam, chlordiazepoxide, N-desmethyldiazepam, N-desmethylchloridazepoxide, and demoxepam in serum by high performance, liquid chromatography.

A method is presented for simultaneously determining diazepam and chlordiazepoxide along with their respective major active serum metabolites N-desmethyldiazepam, and N-desmethylchlordiazepoxide and demoxepam. The drugs are extracted from one ml of buffered serum using chloroform containing 5-(p-methylphenyl)-5-phenylhydantoin as an internal standard. The elution is accomplished using a reversed-phase column with a mobile phase consisting of an acetonitrile/methanol/acetate buffer pH 5.0 (200/225/500) at a flow rate of 2.0 ml/min. Absorbance is monitored at 240 nm using a variable wavelength detector. Each chromatographic separation requires approximately 15 minutes at ambient temperature. Of more than twenty drugs tested for possible interference with this procedure, only methaqualone interferes with the internal standard, and phenytoin with demoxepam.

On the difference between colonic and small intestinal alkaline phosphatase.

Colonic and small intestinal alkaline phosphatase extracts were studied biochemically and electrophoretically to elucidate the source of a reported difference in cellulose acetate electrophoretic mobility. Both preparations were inactivated with 0.5 mmol/L L-phenylalanine but retained full activity in the presence of 1.0 mmol/L tetramisole. Treatment with neuraminidase changed a minor fraction of the small intestinal but the major portion of the colonic alkaline phosphatase to a cathodically migrating form. The most likely explanation for our findings is that the colon and small intestinal alkaline phosphatase are mixtures of the same multiple forms but in different proportions.

Evaluation of the Boehringer Mannheim ES 300 immunoassay analyzer and comparison with enzyme immunoassay, fluorescence polarization immunoassay, and radioimmunoassay methods.

The ES 300 (Boehringer Mannheim Diagnostics, Indianapolis, IN) is a new automated immunoassay analyzer intended for the quantitative determination of a wide range of analytes. We compared its performance to enzyme immunoassay (EIA), fluorescence polarization immunoassay (FPIA), and radioimmunoassay (RIA) methods for cortisol, digoxin, ferritin, prolactin, T4-uptake, total-T3, and TSH. The ES 300 methods showed excellent precision and the manufacturers' linearity claims were met in all cases. Cortisol, prolactin, total-T3, and TSH showed no bias and acceptable correlation with other methods. Digoxin, ferritin and total T4 showed positive bias but acceptable correlation. The ES 300 T4-uptake correlated poorly with the TDx method and showed positive bias; however, these assays appear comparable (although deficient) in diagnostic sensitivity when compared to TSH and T4 data for the same patient population. In all, we found the ES 300 to be an acceptable instrumental alternative for the high volume immunoassay laboratory.

Chromatographic separation of the C(1), C(1a), and C(2) components of gentamicin and the assessment of their individual binding to serum proteins.

[3H]gentamicin and [14C]gentamicin samples were purified by Sephadex column chromatography and separated by an HPLC technique into the three major, medicinally active gentamicin components. These separated components were used in equilibrium dialysis studies to determine their percent binding to serum proteins. The bindings of the components were inversely related to concentrations of ionized calcium and magnesium. When dialyzed against a buffer containing physiological concentrations of the divalent cations, the binding of the C(1) component was 2.2 +/- 1.0%, the binding of the C(1a) component was 1.2 +/- 1.9%, and the binding of the C(2) component was 5.0 +/- 2.0%. The percent bindings are not identical and, due to their low values, probably have negligible clinical significance. The radioactive composition and purity of the 3H- and 14C-labeled gentamicin samples differed and these may be important factors in the variance of reported gentamicin bindings.

Bis-(2-ethylhexyl) phthalate, an ubiquitous environmental contaminant.

Bis-(2-ethylhexyl) phthalate (DEHP) is the most commonly used plasticizing agent for the widely used plastic polyvinylchloride (PVC). Consequently, this compound is found everywhere in the environment of civilization, where it is in frequent contact with every person. Blood storage bags and tubing, food wrappers, and many children's toys contain appreciable amounts of DEHP. Given this frequency of exposure, the toxic potential of the compound has become a major concern. Many workers have demonstrated its exceedingly low acute toxicity, while results from chronic exposure studies have been mixed. However, in 1982 the National Toxicology Program reported a significantly increased incidence of hepatocellular carcinoma in rats and mice exposed to high doses of DEHP over a period of two years. The significance of these studies remains in question. Bis-(2-ethylhexyl) phthalate is metabolized extensively by mammals, but reports of the direct study of the toxic effects of its metabolites are few. Efficient methods for analysis of biological samples for DEHP are available, but they are complicated by the constant presence of this compound as a contaminant.

Analysis of serum for gentamicin by radioimmunoassay.

A radioimmunoassay method for the determination of combined gentamicin isomers in serum has been adapted and tested. The procedure uses tritiated gentamicin, rabbit antiserum against a gentamicin-human albumin conjugate, and dextran-charcoal separation. The day-to-day coefficient of variation is 6 percent, and frozen samples are stable for at least one month. There was no cross reactivity of the antiserum with any of several tested antibiotics or with any of some tested commonly used non-antibiotic hospital pharmacy preparations. The results from the procedure correlated well with those of an enzymatic radioacetylation technique and, except for a significant incidence of "outliers", with a microbiological assay. As expected, patient serum values, both maximum and minimum, showed no correlation with dose size.

Comparison of a commercial method for total protein with a candidate reference method.

The biuret method for total protein has been compared on the Boehringer Mannheim Diagnostics (BMD) Hitachi 717 Analyzer with a candidate reference method in efforts to standardize the BMD method. The methods were compared using 115 paired serum specimens collected during morning rounds at the Roger Williams Hospital. Results from the test method were compared to the reference method using a statistical procedure for quantifying bias between analytical methods. Linear regression statistics were also calculated. The Hitachi 717 biuret method shows no bias when compared to the reference method (z = -0.90), and there is acceptable correlation between the two methods (y = 0.86, m = 0.86, r = 0.896). The Hitachi method is linear to 15.0 g per dL and demonstrates excellent precision (CV less than or equal to 2.14 percent, N = 140). The Hitachi 717 biuret method has been found to be excellent in all respects and its use is recommended as a convenient and accurate means of measuring total protein.

Comparison of fluorescence polarization immunoassay, enzyme immunoassay, and thin-layer chromatography for urine cannabinoid screening. Effects of analyte adsorption and vigorous mixing of specimen on detectability.

Four commercial assays for the screening of cannabinoids in urine were compared. Urine specimens from 93 selected subjects were run by fluorescence polarization immunoassay on the Abbott TDx; by enzyme multiplied immunoassay with two Syva EMIT assays; and by thin-layer chromatography with the TOXI-LAB system (Marion Laboratories). The TDx cannabinoid threshold can be set anywhere from 25 to 150 micrograms per L. Twenty-five micrograms per L was chosen for this study. The thresholds for EMIT are fixed at 20 micrograms per L for one assay and 100 micrograms per L for the other. The detection limit for TOXI-LAB, according to the manufacturer, can be anywhere from 5 to 50 micrograms/L, depending on the specimen. Urines, positive by at least one method, were further analyzed by gas chromatography with mass spectrometry (GC/MS), The detection limit for the GC/MS method was 10 micrograms per L. The results showed a few false negatives and unconfirmable positives; in general, correlation was considered acceptable. Dose-response curves comparing TDx and EMIT gave paralell results, with comparable cross-reactivity for the major metabolite, 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (delta-9-THC-COOH). A dose-response study of TOXI-LAB using delta-9-THC-COOH also gave acceptable results. Adsorption to glass was investigated using spiked urine; a 27 percent reduction in concentration was caused by this phenomenon. Foaming of spiked urine caused by vigorous mixing resulted in a reversible 89 percent apparent reduction in concentration.

A direct current plasma emission spectrometric procedure for the assay of silicon in urine.

A method for the assay of silicon in urine has been developed using direct current plasma emission spectroscopy. Urine is directly aspirated into the argon plasma, and the silicon emission is measured at 251.6 nm. There is a moderate matrix effect which is not compensated by the addition of lithium chloride as an ionization suppressor. The use of calibration standards in an urine-like matrix gives the best analytical results as documented by serial dilution and standard addition studies. The method is linear over the entire range of urine concentrations normally encountered (0 to 50 mg per L). The lower detection limit is 0.05 mg per L and the coefficients of variation at the 2.7 mg per L and 5.9 mg per L levels are 5.8 percent and 11.5 percent, respectively. Urine from ten randomly chosen volunteers shows a considerable between subject variation in silicon concentration (mean 12.5 +/- 8.3 mg per L, range 1.8 to 51.6 mg per L) which is positively associated with vegetable intake. Urine from 39 hospital patients on a standardized diet shows much less variation in concentration (mean 4.1 +/- 3.2 mg per L, range 0.1 to 18.6 mg per L), indicating that the urine silicon assay is likely to be useful in metabolic studies only if diet is controlled.

Analysis of human serum proteins by molecular weight dependent acrylamide gel electrophoresis.

The technique of acrylamide gel electrophoresis of sodium dodecyl sulfate treated protein mixtures has been applied to the analysis of human serum proteins in the 70,000 to 250,000 molecular weight range. After staining, the bands are well defined, the molecular weight is defined and, hence, the identity of each can be estimated from the migration distance. In ambiguous cases, the identification of a band is confirmed by an independent method. The procedure is particularly valuable in the study of the gammopathies. The immunoglobulins migrate as expected, in contrast to the problems often encountered in the Ornstein-Davis gel method. Furthermore, light chains migrate more rapidly, are well separated from other serum proteins and, therefore, are readily detected. Paraproteins from three patients with documented gammopathies have been studied and characterized using this method.

The significance of beta-2 microglobulinuria associated with gentamicin therapy.

Gentamicin is a nephrotoxic agent known to damage the proximal tubule,--a site of low molecular weight (LMW) protein reabsorption and catabolism. The effect of gentamicin was investigated on three LMW proteins--amylase, light chains, and beta 2 microglobulin--and the effects were correlated on the latter to renal function as determined by creatinine clearance (GFR). The renal excretion of beta 2 microglobulin (beta 2M) was studied in 18 patients receiving gentamicin and eight control patients. Both gentamicin and control patients had similar mean ages and serum beta 2M. Twelve of the 18 gentamicin treated patients had marked increases in beta 2M excretion. The mean daily 2 beta microglobulin excretion for the gentamicin treated group was 10,511 microgram while that of the control group was 102 microgram. Serial determinations in 10 of the gentamicin treated patients revealed an increase in beta 2M excretion within 48 hours of starting therapy. No deterioration of GFR was seen in any patient. In four patients, beta 2M excretion decreased while still receiving gentamicin. The renal handling of amylase was found to be normal in four patients and mildly abnormal in three patients receiving gentamicin who also had increased beta 2M excretion. Urinary light chains were determined in four of these seven patients and found to be normal. It is concluded that gentamicin induces an early and often transient tubular proteinuria. This tubular proteinuria is not associated with clinical nephrotoxicity.

Tissue analysis of N-methylformamide: organ distribution.

This report describes a gas chromatographic procedure, utilizing a packed column and flame ionization detector, suitable for the quantitative measurement of N-methylformamide (N-MF) in tissue samples. N-MF is a polar solvent that induces the maturation of cancer cells in vitro and, in vivo, exhibits antitumor activity with human tumors xenografted in nude (athymic) mice. Therapeutic monitoring is essential as toxicology studies have shown this compound to be hepatotoxic. N-MF is currently undergoing phase 1 clinical trials as an anticancer drug. This method of tissue analysis was developed to aid in the understanding of N-MF disposition and distribution in a murine model. The data thus generated may help predict the clinical behavior of this drug.

A therapeutic monitoring assay for N-methylformamide in human serum and urine.

This report describes a gas chromatographic procedure using a packed column and a flame ionization detector, which is suitable for the therapeutic monitoring of N-methylformamide (N-MF). N-MF is a polar compound that induces cancer cell maturation in vitro and exhibits antitumor activity with human tumors xenografted in nude mice. Toxicology studies with mice have shown this compound to be hepatotoxic. N-MF is currently undergoing Phase I clinical trial as an anticancer drug at this institution. In concert with its clinical trial, a method for the analysis of N-MF in biological fluids has been developed to study its pharmacokinetics. This method involves direct injection of urine or acetone-deproteinized serum onto a Chromosorb 103 column, using N,N-diethylformamide as an internal standard.