Mixed muscle and hepatic derived plasma protein metabolism is differentially regulated in older and younger men following resistance exercise.

We sought to determine whether exercise-induced muscle protein turnover alters the subsequent production of hepatically derived acute-phase plasma proteins, and whether age affects how these proteins are regulated. We measured arteriovenous (a-v) balance and the synthesis of mixed muscle protein, albumin (A) and fibrinogen (F) before exercise (REST) and from the beginning of exercise to 10, 60, and 180 min following a single bout of moderate-intensity leg extension exercise (POST-EX) in postabsorptive untrained older (n = 6) and younger (n = 6) men using L-[ring-2H5]phenylalanine (Phe). Subjects performed 6 sets of 8 repetitions of leg extension at 80% of their 1-RM (one-repetition maximum). All data are presented as the difference from REST (Delta from REST at 10, 60, and 180 min POST-EX). Mixed muscle fractional synthesis rate (FSR-M) increased significantly from the beginning of exercise until 10 min POST-EX in the older men (DeltaFSR-M: 0.044%/h), whereas FSR-M in the younger men was not elevated until 180 min POST-EX (DeltaFSR-M: 0.030%/h). FSR-A and FSR-F increased at all POST-EX periods in the older men (DeltaFSR-A = 10 min: 1.90%/day; 60 min: 2.72%/day; 180 min: 2.78%/day; DeltaFSR-F = 10 min: 1.00%/day; 60 min: 3.01%/day; 180 min: 3.73%/day). No change occurred in FSR-A in the younger men, but FSR-F was elevated from the beginning of exercise until 10 and 180 min POST-EX (10 min: 3.07%/day and 180 min: 3.96%/day). Net balance of Phe was positive in the older men in the immediate POST-EX period. Our data indicate that mixed muscle and hepatic derived protein synthesis is differentially regulated in younger and older men in response to a single bout of moderate-intensity leg extension exercise. Moreover, our data suggest that with age may come a greater need to salvage or make available amino acids from exercise-induced muscle protein breakdown to mount an acute-phase response.

Assessment of 1,3-butadiene exposure in polymer production workers using HPRT mutations in lymphocytes as a biomarker.

1,3-Butadiene (BD), which is used to make styrene-butadiene rubber, is a potent carcinogen in mice and a probable carcinogen, associated with leukemia, in humans. We have previously used HPRT mutation as a biomarker to evaluate exposures to BD in a monomer production plant. We now report on a study of 49 workers in a styrene-butadiene rubber plant in which we used the concentration of the BD metabolite 1,2-dihydroxy-4-(N-acetylcysteinyl-S)-butane (M1) in urine as a biomarker of exposure and the frequency of HPRT variant (mutant) lymphocytes (Vf) as a biomarker of effect. Workers were assigned to high- and low-exposure groups based on historical information about work areas and jobs. Personal exposure to BD for one work shift was measured using a passive badge dosimeter. Each participant provided a urine specimen and blood sample at the end of the work shift and completed a questionnaire providing information on lifestyle, health, and work activities. The average BD exposures in the high- and low-exposure groups were significantly different, even after excluding two extreme values, (high 1.48 ppm; low 0.15 ppm, p < 0.002). This study was done in 1994 and 1995 before the establishment, in 1996, of the new permissible exposure limit of 1 ppm. Both the mean M1 and the HPRT Vf were more than three times greater in the high-exposure group than in the low-exposure group (p < 0.0005). The three end points correlated with each other, with sample correlation coefficients between 0.4 and 0.6. The correlations among BD exposure and the biomarkers of internal exposure and genotoxicity suggest that occupational exposure to BD, in the range of 1-3 ppm, may be associated with adverse biological effects.

Assessment of butadiene exposure in synthetic rubber manufacturing workers in Texas using frequencies of hprt mutant lymphocytes as a biomarker.

1,3-Butadiene (BD), which is used to manufacture synthetic rubber, is a mutagen and carcinogen. Because past occupational exposures have been associated with an increased risk of leukemia, there has been a dramatic reduction in workplace exposure standards. The health benefits of these reduced levels of occupational exposure to BD will be difficult to evaluate using relatively insensitive traditional epidemiological studies; however, biomarkers can be used to determine whether there are genotoxic effects associated with recent exposures to BD. In past studies of BD-exposed workers in Southeast Texas, we observed an increase in the frequency of lymphocytes with mutations in a reporter gene, hprt. Frequencies of hprt mutant cells correlated with air levels of BD and with the concentration of a BD metabolite in urine. Average exposures to 1-3 parts per million (p.p.m.) of BD were associated with a threefold increase in hprt variant (mutant) frequencies (Vfs). We now report results from a follow-up study of workers in a synthetic rubber plant in Southeast Texas. Thirty-seven workers were evaluated on three occasions over a 2-week period for exposure to BD by the use of personal organic vapor monitors and by determining the concentration of a BD metabolite in urine. The frequency of hprt mutants was determined, by autoradiography, with lymphocyte samples collected 2 weeks after the final exposure measurement. Based on their work locations, the study participants were assigned to high-exposure (N=22) or low-exposure (N=15) groups. The BD exposure, +/-standard error, of the workers in the high-exposure group (1.65+/-0.52 p.p.m.) was significantly greater than the low-exposure group (0.07+/-0.03 p.p.m.; P<0.01). The frequency of hprt mutant lymphocytes was also significantly different in the two groups (high, 10.67+/-1.5 x 10(-6); low, 3.54+/-0.6 x 10(-6); P<0.001). The concentration of the urine metabolite was greater in the high-exposure group, but the difference was not significant. The correlation coefficient between hprt Vf and BD exposure levels was r=0.44 (CI(95), 0.11-0.69; P=0.011). This study reproduced the findings from a previous study at this plant. Although studies of butadiene-exposed workers in other countries have not detected an effect of exposure on frequencies of hprt mutant lymphocytes, we have repeatedly observed this result in our studies in Texas.

Molecular analysis of hprt mutant lymphocytes from 1, 3-butadiene-exposed workers.

1,3-Butadiene (BD) has been shown to be a potent animal carcinogen and a probable human carcinogen, yet the molecular mechanisms of BD genotoxicity and carcinogenicity still are not fully understood. Our hypothesis is that metabolites of BD induce specific structural changes in the human hprt gene like those observed in vitro in TK6 cells and in vivo in the mouse. Characteristic mutations in BD-exposed subjects can be identified and used as biomarkers for monitoring genotoxic effects associated with BD exposure. Molecular analysis of hprt mutant lymphocytes from BD-exposed workers and unexposed control subjects was carried out to identify changes in the structure of the hprt gene. A multiplex polymerase chain reaction (PCR) assay was used to detect exon deletions in 360 hprt mutant clones. We determined that exon deletions were significantly more frequent (P < 0.05) in BD-exposed workers (17.5%) than in control subjects (9.7%). Sequence analysis of hprt cDNA from 175 independent mutants indicated that the distribution of the types of mutations was different between the workers and the unexposed control subjects. There was a significant increase in -1 frameshift mutations in BD-exposed workers, predominantly in repeated DNA sequences, and single-base substitutions were decreased to 66% in the workers compared to 83% in the control subjects (P < 0.05). In addition to the spectral changes, hprt clonal assays revealed an elevation in mutant frequency in the lymphocytes of workers (N = 10) when compared with that in unexposed control subjects (N = 11; P < 0. 05). There also was a twofold increase of A:T --> T:A transversions in BD-exposed workers (16% in BD-exposed workers compared to 8% in controls, P = 0.25). Some of the BD-associated changes in mutational spectra observed in our study have the potential for application in monitoring genotoxic effects related to butadiene exposure.

Some theoretical and practical considerations for multivariate statistical cell classification useful in autologous stem cell transplantation and tumor cell purging.

BACKGROUND: As flow cytometric data becomes more complex, it becomes increasingly difficult to classify cells using conventional flow cytometry data techniques based on visual classification of the data by user-drawn regions. This paper shows some simple applications of multivariate statistical classification to classify flow cytometric data. METHODS: Discriminant Function Analysis (DFA) and Logistic Regression (LR) analysis techniques were evaluated with respect to their potential utility in the problem of detecting human breast cancer cells within normal bone marrow cells. Data sets having defined properties were employed to evaluate the potential utility of these statistical classification techniques whose performance was measured by ROC analysis. RESULTS: Two extreme but reasonable situations are presented: (1) data where the separation of cells was obvious by visual inspection and (2) data where major overlaps in the values of the individual FCM parameters made intuitive classification improbable. Both DFA and LR analysis were able to classify the cells of each type with acceptable accuracy and yield. CONCLUSIONS: The excellent empirical performance of both DFA and LR techniques, suggests that they offer promising approaches for classifying multiparameter FCM data using objective rules that may represent an improvement over commonly employed ad hoc approaches.

Kinetics of repair of UV-induced DNA damage in repair-proficient and -deficient cells as determined by quantitative polymerase chain reaction.

Advances in methodologies to monitor gene-specific repair in human cells have facilitated a detailed understanding of the complexity of the nucleotide excision repair system. One of these procedures, quantitative polymerase chain reaction (QPCR), holds significant promise for dissecting the fine structure of the repair of UV-induced DNA damage. This assay was used to study the repair of UV photoproducts in both actively transcribed and nontranscribed genes from human cells that were capable of (1) repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts; (2) removal of neither dimers nor 6-4 photoproducts; (3) strong preferential repair of 6-4 photoproducts relative to dimers; and (4) severely depressed rates of 6-4 photoproducts and dimers. Detailed kinetic analyses revealed that repair of both active and inactive genes can be studied with a very fine degree of precision and that the repair status of the cells can easily be detected by use of the procedures described.

Theoretical basis for sampling statistics useful for detecting and isolating rare cells using flow cytometry and cell sorting.

This paper describes new approaches to calculating the number of cells that need to be processed using flow cytometry (FCM) techniques and the subsequent time required in order to isolate a specific number of cells having selected characteristics. The methods proposed use probabilistic assumptions about the contents of the sample to be sorted, logarithmic/exponential transformations to avert the computer "underflow" and "overflow" limitations of brute force calculations for the parameters of the binomial distribution imposed by existing computer hardware, and an established mathematical procedure for calculating error bounds for the normal approximation to the binomial distribution. Estimates are derived for the total number of cells in the FCM sample volume that must be available for processing and, for given FCM cell sorting decision speeds, the total elapsed times necessary to conduct particular experiments. The proposed approach obviates the need to resort to calculation expediencies such as the theoretically limited Poisson approximation for what can be considered a Bernoulli process mathematically characterized by the binomial distribution. Tables and graphs illustrate the projected times required to complete FCM experiments as a function of "effective" cell sorting decision speeds. Results from this paper also demonstrate that, as the "effective" cell sorting decision speed increases, there may not be a corresponding linear decrease in the time required to sort a given number of cells with selected statistical properties. The focus of this paper is on the use of innovative mathematical techniques for the design of experiments involving rare cell sorting. However, these same computational approaches may also prove useful for the high-speed enrichment sorting of non-rare cell subpopulations.

A modified quantitative polymerase chain reaction assay for measuring gene-specific repair of UV photoproducts in human cells.

Methods for measuring the induction and repair of ultraviolet (UV) induced modifications in short DNA fragments are essential for the study of gene-specific DNA repair. Measurements in genomic fragments of 14 kilobases (kb) or larger can be obtained using the enzyme-sensitive site (ESS) assay introduced by Hanawalt and Bohr (Bohr et al., 1985). More recently, several assays based on variations of the polymerase chain reaction (PCR) technique have been developed which have increased sensitivity (Govan et al., 1990; Kalinowski et al., 1992; Jennerwein and Eastman, 1991), even nucleotide resolution (Pfeifer et al., 1993). However, examination of these reports indicates that the PCR based DNA repair assays lack precision (Govan et al., 1990; Kalinowski et al., 1992; Tornaletti and Pfeifer, 1994; Jennerwein and Eastman, 1991). We report here, the development of a highly precise QPCR DNA repair assay. The assay was used to measure the induction and repair of UV photoproducts in a 2.7 kb genomic fragment containing the human growth hormone (hGH) gene in SL89 (wild-type) fibroblasts. The assay was exceedingly reproducible with an overall coefficient of variation from the mean of about 2.5%. This level of precision enabled the apparent simultaneous resolution of cyclobutane dimer (CPD) and (6-4) photoproduct (6-4PP) induction and repair.

Effect of dichloroacetate on lactate concentration in exercising humans.

The precise mechanism responsible for the increase in plasma lactate concentration during exercise in humans is not known. We have used dichloroacetate to test the hypothesis that a limitation in pyruvate dehydrogenase activity is responsible for the rise in plasma lactate. Dichloroacetate stimulates the activity of pyruvate dehydrogenase, which is normally the regulatory enzyme in the oxidation of glucose when tissue oxygenation is adequate. Six subjects were studied twice according to a randomized, crossover protocol, involving one test with saline infusion and another with dichloroacetate infusion. Exercise load on a bicycle ergometer was increased progressively until exhaustion. Blood samples were drawn each minute throughout exercise and periodically throughout 120 min of recovery. Dichloroacetate significantly lowered the lactate concentration during exercise performed at less than 80% of the average maximal O2 consumption. The peak concentration of lactate at exhaustion was not affected by dichloroacetate treatment, but dichloroacetate did lower lactate concentration throughout recovery. These results suggest that a limitation in pyruvate dehydrogenase activity contributes to the increase in plasma lactate during submaximal exercise and recovery.

Total cerebral ischemia: effect of alterations in arterial PCO2 on cerebral microcirculation.

Radiolabeled 15-microns microspheres were used to examine alterations in regional CBF and cerebrovascular resistance in response to changes in arterial PCO2. Flow measurements were obtained before and 1-3 and 24 h after 12 min of total cerebral ischemia. Striking sensitivity of blood flow in all areas of the central nervous system was shown to changes in arterial PCO2 between 24 and 50 mm Hg during the control nonischemic period. Following 12 min of total cerebral ischemia, cerebrovascular resistance increased, producing a decrease in regional blood flow when the important controlling variables for CBF were held constant. One to 3 h after total cerebral ischemia, the effect of variations in arterial PCO2 on cerebral blood flow was almost completely abolished. Within 24 h after total cerebral ischemia, the sensitivity of CBF to changes in PCO2 was almost completely restored, whereas the secondary severe neurologic deficit remained. Therapeutic interventions following global cerebral ischemia, designed to ameliorate the "no-reflow" phenomenon and minimize residual ischemic neurologic damage, must take into account this marked early post-ischemic reduction in sensitivity to normally potent cerebrovasodilatory influences.

Effect of thiopental therapy on cerebral blood flow after total cerebral ischemia.

The effect of barbiturate coma upon regional cerebral blood flow (RCBF) and ultimate neurologic outcome was examined after total cerebral ischemia (TCI). TCI was induced in dogs using a relatively noninvasive double-occlusion balloon technique; cardiopulmonary protection was provided during the period of ischemia. RCBF was measured using 15-mu radioactively labeled microspheres. A reproducible pattern of impaired reperfusion of the central nervous system (CNS) was observed in control animals after the restoration of cerebral perfusion pressure after TCI. This pattern was accentuated by the administration of pentothal to induce barbiturate coma. The additional depression in RCBF in those animals receiving pentothal was most prominent in cortical gray matter and brainstem structures at 3 and 6 h after TCI. It was also observed in cortical white matter. No untreated animal surviving TCI achieved a neurologic functional level better than persistent vegetative (decerebrate) survival over 1 wk of observation. Animals receiving 90 mg/kg body weight of pentothal post-TCI demonstrated irreversible cardiogenic shock related to the myocardial depressant effect of the drug. Animals receiving 40 to 60 mg/kg of pentothal post-TCI demonstrated a survival rate similar to that of untreated animals. Although this study did not establish the possible effectiveness of barbiturate coma in improving residual neurologic damage after TCI, the data do demonstrate that any possible effectiveness in this model is not associated with any improvement in the markedly decreased cerebral perfusion after TCI.

Relative error and variability in blood flow measurements with radiolabeled microspheres.

The validity of blood flow measurements made using radiolabeled microspheres (reference sample technique) has been well established in a variety of experimental models. Previous studies of the variability of the method emphasize tissue sphere number as the principle source of random variation. However, blood flow measurements depend on sphere distribution to both tissue and reference samples as well as on quantitation of different isotopes in a single tissue sample. In this study we examined the major determinants of relative error and variability in flow rate measurements inherent in the reference sample technique. Error magnitude was predicted from a statistical model, simulated with numerical analogs, and measured in anesthetized dogs with 15-micrometers microspheres. Theoretically, with 2,000 spheres in the reference sample and greater than or equal to 475 spheres in a tissue sample, blood flow can be measured with 10% accuracy at the 95% confidence level and with duplicate variability of 14%. Random errors in isotope quantitation were influenced by the specific activity of the isotope and the fractional distribution of a given isotope over the various energy windows. Suspension of tissue and reference sample spheres at different heights (0.2 cm) resulted in systematic flow errors of up to 13%. When errors due to separation of isotopes and differences in sample height were minimized, the major determinant of variability in flow measurements during simultaneous injection of differently labeled microspheres was the number of spheres in both tissue and reference samples.

Barbiturate-induced coma therapy for focal cerebral ischemia. Effect after temporary and permanent MCA occlusion.

The authors have studied the therapeutic effect of barbiturate coma following middle cerebral artery (MCA) occlusion in primates. The relationship of the efficacy of barbiturate protection to the presence or absence of recirculation was examined. Barbiturate therapy was begun 30 minutes after MCA occlusion. The findings were as follows: 1) barbiturate-induced coma, with its attendant monitoring, was safely tolerated by primates for 96 hours; 2) 6 hours of MCA occlusion followed by recirculation resulted in a neurological deficit that was worse than the neurological deficit produced by permanent MCA occlusion; 3) barbiturate-induced coma for 96 hours, initiated 30 minutes after the onset of MCA occlusion, in the absence of reperfusion, was in fact detrimental; 4) barbiturate-induced coma for 96 hours, initiated 30 minutes after MCA occlusion, with the establishment of reperfusion at 6 hours, provided nearly complete protection from ischemic damage.

Total cerebral ischemia: application of a new model system to studies of cerebral microcirculation.

Reduction in cerebral blood flow (CBF) following global ischemia has been implicated as a pathogenetic mechanism in progressive brain damage seen after restoration of effective cardiac action and cerebral perfusion pressure. There are serious limitations to many of the techniques for measuring regional cerebral blood flow, particularly during low flow states. In 15 dogs anesthetized with thiopental, 12 minutes of total cerebral ischemia (TCI) was produced using a double balloon occlusion technique. Total and regional cerebral blood flows were sequentially measured before and after balloon release by left ventricular injection of 15 mu microspheres labelled with 5 different radionuclides. Total CBF was reduced 53 +/- 5% (mean +/- SEM) from pre-ischemic values between 1 and 3 hours after "resuscitation" despite normal perfusion pressure and arterial blood gases. CBF remained slightly reduced (24 +/- 7%) at 6 hours post-ischemia. Thirty minutes after balloon release, grey matter flow was reduced 38 +/- 8% from control values while adjacent white matter flow was increased 21 +/- 10%. However, by 1 hour after ischemia, grey and white matter flows were both reduced (60 +/- 3%, 41 +/- 5% respectively). Similar differences in brain stem and cerebellar flow were also observed. The majority (71-86%) of the reduction in total CBF seen at one hour post-TCI is due to increased cerebrovascular resistance, with 14-29% of the decrease related to arteriovenous shunting.