Problems in determination of skeletal lead burden in archaeological samples: an example from the First African Baptist Church population.

Human bone lead content has been demonstrated to be related to socioeconomic status, occupation and other social and environmental correlates. Skeletal tissue samples from 135 individuals from an early nineteenth century Philadelphia cemetery (First African Baptist Church) were studied by electrothermal atomic absorption spectrometry and X-ray fluorescence for lead content. High bone lead levels led to investigation of possible diagenetic effects. These were investigated by several different approaches including distribution of lead within bone by X-ray fluorescence, histological preservation, soil lead concentration and acidity as well as location and depth of burial. Bone lead levels were very high in children, exceeding those of the adult population that were buried in the cemetery, and also those of present day adults. The antemortem age-related increase in bone lead, reported in other studies, was not evidenced in this population. Lead was evenly deposited in areas of taphonomic bone destruction. Synchrotron X-ray fluorescence studies revealed no consistent pattern of lead microdistribution within the bone. Our conclusions are that postmortem diagenesis of lead ion has penetrated these archaeological bones to a degree that makes their original bone lead content irretrievable by any known method. Increased bone porosity is most likely responsible for the very high levels of lead found in bones of newborns and children.

PCR libraries of ancient DNA using a generalized PCR method.

We describe a generalized PCR method that will amplify fragments of DNA without any knowledge of sequence using a single primer. Although we are presently using this method to amplify DNA fragments isolated from ancient preserved tissues, in effect, producing PCR libraries, it may prove to have other applications.

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease.

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing. Hybridization screening of the covalently immobilized PCR products positively identified products from 25 of 27 specimens of different tissues from these four mummies. The method appears to be ideal for the purpose of screening a large number of specimens when the target PCR product is very short.

Preserved human remains from the southern region of the North American continent: report of autopsy findings.

Autopsies were performed in six naturally mummified bodies from the southern area of North America. The cause of death was established with reasonable certainty in two (abdominal stab wound and sand pneumoconiosis); was somewhat speculative in one (atelectasis secondary to bronchial obstruction by an aspirated tooth); and was undetermined in three. The value of such studies is discussed, and the synergistic effect of interdisciplinary contributions is emphasized.

Lead in bone. I. Direct analysis for lead in milligram quantities of bone ash by graphite furnace atomic absorption spectroscopy.

A method for direct lead content analysis of milligram quantities of bone ash by flameless atomic absorption spectroscopy is described. Bone ash (25 mg) is dissolved with HNO3 and diluted with H2O and La2O3 (1,000 micrograms/ml) solution. Lanthanum ion is used to suppress matrix interferences possibly arising in part from sulfate components of the bone ash. Two bulk bone samples (about 14 and 60 micrograms Pb/g ash, respectively) were used to determine daily, within-day, and overall variability of the method. Values for "low lead" bone samples were 14.08 +/- 1.74 (SD) microgram Pb/g ash and for "high lead" bone samples were 60.85 +/- 5.24 (SD) microgram Pb/b ash. The overall value of 58 lead recovery determinations from bone ash analysis was 103.5% (+/- 12.9% SD). These values compare favorably with results previously reported using gram amounts of sample.

Protein oxidation of a hair sample kept in Alaskan ice for 800-1000 years.

Ancient finds of organic matter are not only of the highest value for palaeochemists and palaeobiologists but can be used to determine basic chemical reactions, such as protein oxidation, over long time periods. We studied oxidation of human hair protein about one thousand years old of an Alaskan child buried in ice, ten hair samples of copts of comparable age buried in graves of hot dry sand and compared the results to ten recent hair samples. Protein oxidation parameters o-tyrosine and cysteic acid of the Alaskan child were comparable to recent samples whereas they were higher in the coptic specimen. N-epsilon-carboxymethyllysine, a parameter for glycoxidation, however, was as high in coptic specimen. We conclude that ice in contrast to soil prevented protein oxidation but failed to inhibit glycoxidation, a reaction initiated by autooxidation of glucose. This study therefore has implications for the interpretation of oxidation and glycoxidation as well as preservation mechanisms of proteins.

Selected aspects of the spatial distribution of lead in bone.

The macrodistribution of lead within the skeletal system is not uniform, and is clearly dependent on bone type (compact or trabecular), age and (to a lesser extent) gender. During active growth, lead deposition favors trabecular bones sites but in adulthood lead accumulates more actively in compact bone. The extent and patterns of the higher variability in trabecular bone sites require better definition through further study. Lead tends to be deposited in bone at sites of most active calcification at the time it is absorbed. During the first 15 years of life the bone growth centers dominate the process of calcification. In adulthood osteon remodelling and trabecular plate mineral exchanges are the principal calcifying sites. Both patterns probably deteriorate in old age. Microdeposition patterns of lead could be expected to conform to these changes. However, it is difficult to integrate the reported information into a clearly defined chronological sequence, because many of the studies did not control for age and other variables with sufficient precision. Furthermore, kinetic studies suggest lead is deposited in multiple sites not only among soft tissues but also within bone in molecular forms possessing varying affinities. Current efforts to design instrumentation for in vivo diagnostic lead measurements as well as therapeutic methods to mobilize and remove lead in bone require more precise knowledge than is now available concerning lead macrodistribution, microdistribution and anatomic sites of kinetic compartments. Appropriately designed studies employing contemporary, sensitive techniques involving isotopes, microradiographs, autoradiographs, histology, tomography, and high energy x-ray fluorescence (synchrotron radiation-induced x-ray emission-SRIXE) are now capable of generating the needed data.

Comparison of two in vitro methods of bone lead analysis and the implications for in vivo measurements.

Atomic absorption spectrometry and x-ray fluorescence have been used to determine the lead content of metatarsal and tibia bone samples. For a range of bone lead levels from 6.5 to 83 micrograms g-1 of ashed bone there is no evidence of a systematic difference between the two techniques of more than 1 microgram g-1. There is, however, some evidence that random differences between the two in vitro analyses applied to the same bone sample are larger than can be accounted for by known measurement uncertainties. Variations in bone composition could account for these differences. Because the x-ray fluorescence technique is applied in an identical way to in vivo analysis, it is concluded that the uncertainties in in vivo measurements are small.

Granulomatous coronary arteritis (Takayasu's disease).

The pathologic findings in two patients who died of nonspecific, granulomatous, coronary arteritis (Takayasu's disease) were studied. In one patient, the disease involved the aortic base, with extension into the aortic valve, coronary arteries, and interventricular septum, causing sudden death. Segmental involvement of the abdominal aorta produced an aneurysm. The other patient showed proximal coronary-artery involvement, with severe stenosis and resulting acute myocardial infarction. Newly developed lesions were found in the splenic and renal arteries. The aortic arch was not involved in either patient.

The paleopathology of an Aleutian mummy.

A multidisciplinary team examined an Aleutian mummy from the collection of the Peabody Museum of Archeology and Ethnology of Harvard University, Cambridge, Mass. The mummy, dating from the early 18th century, was of a middle-aged woman who had suffered from pulmonary and ear infections, atherosclerosis, pediculosis, and degenerative joint disease. Another finding was anthracosis, common in ancient bodies and related to indoor heating and cooking fires. Skeletal lead was not found, in contrast with the high levels seen in modern persons. No neoplasms were identified, again consistent with the results of previous studies of ancient human remains. Such comparisons of ancient and modern morbidity and mortality provide a historical perspective on the evolution and cause of human disease.

Cocaine and metabolites in the hair of ancient Peruvian coca leaf chewers.

Cocaine and its metabolites, benzoylecgonine (BZE) and ecgonine methylester (EME), were found in hair samples from ancient Peruvian coca-leaf chewers dating back to AD 1000. Hair was analyzed by gas chromatography/mass spectrometry (GC/MS) to quantitate the concentrations. The two metabolites were found in higher concentration than the parent drug. The metabolite levels appear to be below that of modern cocaine abusers. Gender does not appear to be a factor in the incorporation of drug into hair.

Preservation of human tissue immersed for five years in fresh water of known temperature.

Two human bodies were recovered from the waters of the Duluth, Minnesota harbor. Extensive adipocere formation resulted in remarkable preservation of gross anatomic features of internal organs. Total time of immersion could be precisely estimated at five years. Water temperature during those five years could also be accurately estimated by direct measurements taken during the year following recovery of the bodies and from information supplied by a local electric power generating company. Immersion occurred at the time of the year when water temperature was highest (70 degrees F [21 degrees C]) facilitating the rapid formation of adipocere. A proposed mechanism for formation of adipocere is described.

Lead in bone. IV. Distribution of lead in the human skeleton.

Flameless atomic absorption spectroscopy was used to measure lead concentrations in samples from 5 selected human skeletal sites (tibia, skull, rib, ilium, and vertebra) obtained from 134 hospital autopsies. Lead was distributed unequally among the different bones in distinct patterns that were age-, and to some extent, sex-dependent. To estimate lead concentration of the entire skeleton, all skeletal bones were divided into 5 groups based on their approximate compact/trabecular bone ratios, considering each of our 5 sampled sites to be the prototype for each such group. Regression analysis of the 10 possible bone site pair values at different ages yielded age-related constants. These constants were incorporated into an equation we developed that can be used both to estimate mean skeletal lead concentration (Pb) of the entire body skeleton and also to predict the lead concentration at any of the other 4 bone sites if any 1 of the 5 is measured. Applications of these data to in vivo bone lead measurements are detailed with respect to selection of the site to be measured, estimation of total skeletal lead burden, anticipated variations or error, and dependence of these factors on age and sex of the sampled population.

Biological effects of ingested amosite asbestos, taconite tailings, diatomaceous earth and Lake Superior water in rats.

Because amphibole fibers were found in Lake Superior and in Duluth municipal water, this study was initiated to evaluate the carcinogenicity of ingested asbestos. Groups of 22-30 Sprague-Dawley rats were fed asbestos and related materials during their lifetime and were examined at autopsy after spontaneous death. Test materials were unfiltered Duluth city tap water, municipal water reservoir sediment suspension, taconite plant tailings, amosite asbestos (a "low-dose" group at 20 mg/rat . day an a high-dose group at 300 mg/rat . day) and diatomaceous earth; a control group drank fiber-free well water or filtered city tap water. Autopsy studies revealed no significant increase in the incidence of malignant tumors in any experimental group compared with that in the control group.

Analysis of protein expression patterns in Barrett's esophagus using MALDI mass spectrometry, in search of malignancy biomarkers.

SUMMARY: In order to detect early changes of malignant degeneration in Barrett's esophagus (BE), and to reduce the cost of surveillance, molecular biomarkers of early malignancy have been sought, with limited success, using genomic and immunohistochemical tools. We postulate that direct analysis of epithelial proteins using mass spectrometry will provide protein profiles capable of identifying patients at high risk of developing malignancy. Our aim is to find transitional protein signals that show a cancer profile within histologically benign BE, which can be used as indicators of early malignant change. Fourteen fresh-frozen, resected esophageal cancer specimens were analyzed using laser capture microdissection and matrix-assisted laser desorption/ionization mass spectrometry. Samples of squamous epithelium, and both benign and malignant Barrett's epithelium, were compared for differences in protein expression. Reliable differentiation of squamous and Barrett's epithelium was demonstrated. A comparison of benign and malignant Barrett's epithelium identified a number of cancer-specific protein peaks that were deletion or expression variations from benign epithelium. In four instances the proteins (7350, 8446, 10850, and 14693) appeared to be early malignant changes in histologically benign BE. Mass spectrometry performed upon fresh-frozen Barrett's epithelium, obtained by laser-capture microdissection, displays reproducible, tissue-specific, protein profiles. Distinct differences are demonstrated between benign and malignant epithelium, some of which appear to be candidate biomarkers of early malignant change. This technique reliably displays cellular protein expression in esophageal epithelium and deserves further study as a tool to identify early malignant degeneration in BE.

Design of a pressurization chamber for recordings of retinal blood flow in the "in vivo eyecup" preparation in the rat.

One major goal in our laboratory is development of an "in vivo eyecup" preparation in the rat. This preparation, which involves removal of the cornea and lens, maintains the normal vascular supply of the retina while allowing introduction of pharmacological agents into the medium that bathes this organ. Since we are interested in measuring retinal blood flow, it is sometimes necessary to repressurize the eye to maintain normal perfusion pressure across the retinal vasculature. The goal of this project was to develop a mechanism that allows repressurization of the in vivo eyecup after removal of the front of the eye. The basic design of this chamber consists of an eye ring to mechanically stabilize the eye and a pressurization chamber that attaches to this eye ring. The eye ring is connected to a base via a double hinged arm and can be locked in place. The pressurization chamber is attached to the top of this ring and sealed using an O-ring with a pressure fit. The top of the pressure chamber is composed of a clear polymer, allowing for photic stimulation of the retina, as well as introduction of laser light for blood flow determination. The pressure within the chamber is regulated by an elevated fluid-filled reservoir.