Comparison of an enzyme-linked immunosorbent assay (ELISA) to gas chromatography (GC)--measurement of polychlorinated biphenyls (PCBs) in selected US fish extracts.

The analysis of PCBs in fish tissues by immunoassay methods was evaluated using fish collected from a US monitoring program, the National Contaminant Biomonitoring Program of the US Department of Interior, Fish and Wildlife Service. Selected composite whole fish samples, which represented widely varying concentrations and sources of PCBs, were extracted and subjected to congener PCB analysis by gas chromatography (GC) and total PCB analysis using an ELISA (ePCBs) calibrated against technical Aroclor 1248. PCB congener patterns in these fishes were different from the patterns found in commercial Aroclors or their combinations as demonstrated by principal component analysis of normalized GC congener data. The sum of the PCB congeners measured by GC (total-PCBs) ranged from 37 to 4600 ng/g (wet weight). Concentrations of PCBs as determined by the ELISA method were positively correlated with total-PCBs and the ePCBs/total-PCBs ratios for individual samples ranged from 1 to 6. Ratios of ePCBs/total-PCBs for dilutions of Aroclors 1242, 1254, and 1260 and for matrix spikes range from 0.6 for 1242 to 2.5 for 1254 and 1260. These results suggest that higher chlorinated PCB congeners have higher affinity for the anti-PCB antibodies. Partial least squares with latent variable analysis of GC and ELISA data of selected Aroclors and fish samples also support the conclusion that ELISA derived PCB concentrations are dependent on the degree of chlorination.

Organochlorine contaminants in double-crested cormorants from Green Bay, WI: I. Large-scale extraction and isolation from eggs using semi-permeable membrane dialysis.

A 41.3-kg sample of double-crested cormorant (Phalacrocorax auritus) egg contents was extracted, yielding over 2 L of egg lipid. The double-crested cormorant (DCC) egg extract, after clean-up and concentration, was intended for use in egg injection studies to determine the embryotoxicity of the organic contaminants found within the eggs. Large-scale dialysis was used as a preliminary treatment to separate the extracted contaminants from the co-extracted sample lipids. The lipid was dialyzed in 80x5 cm semi-permeable membrane devices (SPMDs) in 50-ml aliquants. After the removal of 87 g of cholesterol by freeze-fractionation, the remaining lipid carryover (56 g) was removed by 100 routine gel permeation chromatography (GPC) operations. A 41,293-g sample was thus extracted and purified to the extent that it could easily be placed at a volume of 5 ml, the volume calculated to be necessary for the egg injection study. Analyses were performed comparing contaminant concentrations in the final purified extract to those present in the original egg material, in the extract after dialysis and cholesterol removal, and in the excluded materials. Recoveries of organochlorine pesticides through dialysis and cholesterol ranged from 96% to 135%. Total polychlorinated biphenyls in the final extract were 96% of those measured in the original egg material. Analysis of excluded lipid and cholesterol indicated that 92% of the polychlorinated dibenzo-dioxins and -furans were separated into the final extract.

Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.

Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.

Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions.

This study was designed to determine the effects of various environmental factors on the deoxyribonucleic acid (DNA) obtained from dental pulp. Extracted teeth were subjected to the following conditions: varying pH (3,7,10); temperature (4 degrees C, 25 degrees C, 37 degrees C, incineration); humidity (20%, 66%, 98%); various types of soil (sand, potting soil, garden soil); seawater; burying the teeth outdoors, and aging (one week to six months). In addition, teeth that had been extracted and held at room temperature for 16 and 19 years were also examined. Following isolation of DNA, the samples were analyzed on yield gels to determine the concentration and integrity of the recovered DNA. Restriction digestion with Pst I was followed by electrophoresis of the generated fragments, Southern transfer to nylon membranes, and hybridization to both human and bacterial probes. It was determined that, aside from soil, the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. Restriction fragment length polymorphic (RFLP) analysis of selected samples was performed. Dental pulp patterns were compared with bloodstain exemplars, revealing matching patterns, although an increase in band-shifting was observed with extended exposure to elevated temperatures.

Determination of monocyclic and polycyclic aromatic hydrocarbons in fish tissue.

An analytical method is presented in which fish tissue is analyzed for neutral monocyclic and polycyclic aromatic hydrocarbons (AHs) and aromatic sulfur heterocycles (ASHs) by capillary column gas chromatography (CGC) with photoionization detection. The sample enrichment procedure includes saponification with aqueous KOH, acidification of the digestates, and extraction of the aromatic compounds into cyclopentane-dichloromethane. Adsorption chromatography on tandem segments of potassium silicate and silica gel removes 99% of the coextracted lipid. Final enrichment by gel permeation chromatography eliminates residual biogenic material and potentially interfering alkanes. Relatively volatile monoaromatics are included among the analytes by virtue of the efficiency of the complementary enrichment steps, the use of small quantities of only low-boiling solvents, and the selectivity of the detector. Most targeted compounds (AHs ranging in size from C3-alkylbenzenes through benzo[g,h,l]perylene and ASHs within the same size range) can be determined in 5 g (wet weight) samples of fish tissue at concentrations as low as 20 ng/g. Comparisons are made of recoveries of selected AHs under ordinary and gold fluorescent lighting conditions.

Sixty-day infusions of somatotropin-releasing factor stimulate milk production in dairy cows.

Twenty-four Holstein cows were infused intravenously with sterile water (placebo) or 1,3, or 12 mg of recombinant bovine somatotropin-releasing factor 1-45-homoserine lactone/d for 60 d. Relative to placebo (22.8 kg/d), 3 and 12 mg of somatotropin-releasing factor increased yield of milk to 28.8 and 33.3 kg/d during infusion. At 1 mg of somatotropin-releasing factor, milk averaged 27.5 kg/d during infusion but was increased above placebo only through 39 d. After infusion of 12 mg somatotropin-releasing factor ended, milk (26.4 kg/d) remained above placebo amounts (20.6 kg/d) for 15 d. Three and 12 mg of somatotropin-releasing factor increased serum somatotropin from .7 (placebo) to 8.2 and 10.3 ng/ml when averaged across 1, 30, and 59 d, whereas 1 mg increased somatotropin to 5.8 ng/ml after 1 d but had no effect at 30 or 59 d. Within 17 h of cessation of somatotropin-releasing factor infusion, serum concentrations of somatotropin were similar across all groups. On d 59, 3 and 12 mg infusions increased insulin-like growth factor I from 115.8 (placebo) to 204.7 and 261.4 ng/ml of serum. We conclude that somatotropin-releasing factor increased serum concentrations of somatotropin and milk yield in a dose-dependent manner for at least 60 d. Also galactopoietic effects of somatotropin-releasing factor persisted for 15 d independent of increased concentrations of somatotropin in serum following withdrawal of somatotropin-releasing factor.

Microcontaminants and reproductive impairment of the Forster's tern on Green Bay, Lake Michigan--1983.

For the 1983 nesting season, Forster's tern (Sterna forsteri) reproductive success was significantly impaired on organochlorine contaminated Green Bay, Lake Michigan compared to a relatively uncontaminated inland location at Lake Poygan, Wisconsin. Compared with tern eggs from Lake Poygan, eggs from Green Bay had significantly higher median concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), other polychlorinated dibenzo-p-dioxins (PCDDs), total polychlorinated biphenyls (PCBs), total (three congeners) non-ortho, ortho' PCBs, five individual PCB congeners known to induce aryl hydrocarbon hydroxylase (AHH) and several other organochlorine contaminants. Conversions of analytical concentrations of TCDD and PCB congeners based on relative AHH induction potencies allowed for estimation of total 2,3,7,8-TCDD equivalents. Two PCB congeners, 2,3,3',4,4'- and 3,3',4,4',5-pentachlorobiphenyl (PeCB) accounted for more than 90% of the median estimated TCDD equivalents at both Green Bay and Lake Poygan. The median estimated TCDD equivalents were almost 11-fold higher in tern eggs from Green Bay than in eggs from Lake Poygan (2175 and 201 pg/g). The hatching success of Green Bay sibling eggs from nests where eggs were collected for contaminant analyses was 75% lower at Green Bay than at Lake Poygan. Hatchability of eggs taken from other nests and artificially incubated was about 50% lower for Green Bay than for Lake Poygan. Among hatchlings from laboratory incubation, those from Green Bay weighed approximately 20% less and had a mean liver weight to body weight ratio 26% greater than those from Lake Poygan. In both field and laboratory, mean minimum incubation periods were significantly longer for eggs from Green Bay compared to Lake Poygan (8.25 and 4.58 days, respectively). Mean minimum incubation time for Green Bay eggs in the field was 4.37 days longer than in the laboratory. Hatchability was greatly improved when Green Bay eggs were incubated by Lake Poygan adults in an egg-exchange experiment, but was sharply decreased in Lake Poygan eggs incubated in Green Bay nests. Nest abandonment and egg disappearance were substantial at Green Bay but nil at Lake Poygan. Thus, not only factors intrinsic to the egg, but also extrinsic factors (parental attentiveness), impaired reproductive outcome at Green Bay. The epidemiological evidence from this study strongly suggested that contaminants were a causal factor. AHH-active PCB congeners (intrinsic effects) and PCBs in general (extrinsic effects) appeared to be the only contaminants at the concentrations measured in eggs, capable of producing the effects that were observed at Green Bay.