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The ratio of urinary 6beta-hydroxycortisol:cortisol is a measure of the activity of cytochrome p450 3A4 (CYP3A4). CYP3A4 catalyzes the formation of the genotoxic estrogen, 16alpha-hydroxyestrone. It is also involved in the activation of many other mammary carcinogens, such as the polycyclic aromatic hydrocarbons and heterocyclic amines. We evaluated the association between urinary cortisol ratios and breast cancer risk in a subgroup of women who participated in a population-based case-control study in Shanghai. Overnight urine samples from 246 case-control pairs were assayed for 6beta-hydroxycortisol (6beta-OHC) to cortisol. The urine samples from all of the breast cancer patients were collected before any chemotherapy or radiotherapy. In-person interviews were conducted to obtain comprehensive information on dietary habits, reproductive history, and other lifestyle factors. The median levels of 6beta-OHC:cortisol ratios were 2.61 in cases and 2.16 in controls, a 20.8% difference (P < 0.001). The case-control difference was larger in women over 45 years of age (31.3% difference; P < 0.001) than younger women (6.0%; P = 0.45). After adjusting for confounding variables, the risks of breast cancer were increased from 1.0 (reference) to 1.6 [95% confidence interval (CI), 0.9-3.1], 2.2 (95% CI, 1.1-4.2), and 3.7 (95% CI, 1.9-7.4; P for trend, <0.001) with increasing levels of 6beta-OHC:cortisol ratios. The positive association was more pronounced among older women (>45 years) than among younger women (< or = 45 years). The adjusted odds ratios associated with the highest cortisol ratio were 6.0 (95%CI, 2.2-16.1) among older women and 2.2 (95%CI, 0.8-6.1) among younger women. The association of the 6beta-OHC:cortisol ratio was stronger among older women who had a high body mass index, late age at menopause, and early age at menarche (factors related to high endogenous estrogen exposure) than those who did not have these factors. These findings are consistent with the role of CYP3A4 in estrogen and carcinogen metabolism and suggest that high CYP3A4 activity may be a risk factor for breast cancer risk.

The objective of the study was to develop a valid and reliable classification system for failed hip arthroplasties. The study uses research principals derived from multi-attribute utility theory and consensus group techniques. The development of the severity measure was done in two phases. Phase I of the study included: (a) questionnaire development, (b) submission of the questionnaire to the respondents, (c) data synthesis of the responses and item reduction, and (d) classification development and inter-observer reliability testing. Phase II included: (a) resubmission of the instrument to the respondents for suggestions/feedback, (b) instrument revision by the co-investigators based on the respondents' second feedback, and (c) inter-observer reliability testing and intraoperative validity testing of the instrument. The questionnaires sought to capture expert opinion as to what clinical determinants obtained preoperatively (during patient interview, physical exam and review of plain radiographs - AP pelvis and hip lateral) that would in their clinical experience reveal intraoperative severity. There was an 80% (16/20) response rate from the outside experts invited to participate in the study. Based on item reduction and test retest analysis, a five-grade radiographic classification for the acetabulum as well as the femur was developed. This system was then reviewed by 13 of the initial outside experts (16, 80%) who participated in the first round. Inter-rater reliability testing of the final format of the classification revealed a weighted kappa statistic value of 0.88 between the two-blinded raters (inter-rater reliability) and 0.87 between the blinded raters and the reference standard (intraoperative validity). We conclude that the study developed a reliable and valid radiographic classification system for failed hip arthroplasty.

The plasma reduced glutathione (GSH) selenoperoxidase is a highly conserved enzyme. Furthermore, a small clinical study reported that patients with severe atherosclerosis had low peroxidase activities. Together these observations suggest that the peroxidase is important in preventing atherosclerosis. Yet others have reported that when the assay was run in Tris buffer, it was inactive with the concentrations of GSH found in the plasma. Second, it is known that hyperhomocysteinemia increases the rate of atherogenesis. Because there is some homology between homocysteine and the cysteine in GSH, the question is whether the hyperhomocysteinemia effect may be due to inhibition of the peroxidase. We purified the peroxidase from human plasma and determined its activity by a coupled spectrophotometric assay and a substrate disappearance chemiluminescence assay. When the peroxidase activity was determined in phosphate-buffered saline solution (PBS), there was significant activity with the reported plasma GSH concentrations (5 to 20 micromol/L). The peroxidase was exclusively in the HDL fraction. There was no correlation between the peroxidase activity and the HDL or LDL cholesterol concentrations. Finally, at physiologic concentrations of GSH (9 micromol/L), the peroxidase was inhibited by physiologic, free homocysteine concentrations (1 to 5 micromol/L). These data suggest that the peroxidase is active in vivo and may be important in protecting the endothelium from atherosclerosis by preventing oxidant injury. The homocysteine inhibition of the peroxidase suggests a possible biochemical basis for the observed association between hyperhomocysteinemia and cardiovascular disease. Our studies imply that low concentrations of this peroxidase may be an independent risk factor for atherosclerosis.

We previously reported that methoxychlor administration inhibits the activity of the hepatic, microsomal iodothyronine 5'-deiodinase, form I (ID-I; ). Our data further suggested that the inhibition was due to the covalent binding of a methoxychlor metabolite to a 56-kDa protein identified as ID-I (; ). This protein is 98% homologous to the thiol:protein disulfide oxidoreductase, form Q5 (ERp55;; ). Although at the time there was some controversy, most studies now suggest that ID-I is actually catalyzed by a 27-kDa selenoprotein that does not form adducts with methoxychlor (;; ). Because the 27-kDa protein is considered to be ID-I instead of ERp55, we have further examined the basis for the decreased ID-I activity observed after methoxychlor administration. Male, 150- to 200-g Sprague-Dawley rats were given methoxychlor (0-100 mg/kg/day) in corn oil by gavage for 14 days. ID-I was determined by a thyronine-specific immunoassay. Treated rats showed a significant 15% decline in total hepatic, microsomal protein at all doses. The ID-I-specific activity showed a linear decrease with increasing log doses of methoxychlor. The maximum decrease was 42% at 100 mg/kg/day. The 27-kDa protein specific content declined 37%. In rats given methoxychlor the ratios of the 27-kDa protein mRNA to the 18S ribosomal RNA declined from 2.2 +/- 0.27 x 10(-3) (controls) to 0.99 +/- 0.09 x 10(-3) (100 mg/kg/day). These data suggest that the decreased ID-I observed with chronic methoxychlor administration was due to decreased transcription or stability of the mRNA encoding the 27-kDa protein.

The ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) has been suggested as a potential biomarker for breast cancer risk. We evaluated within-person variability of this biomarker in ten healthy Caucasian women aged 23-58 years. Each study participant was asked to provide an overnight fasting morning urine sample once a week for an average of 8 weeks. These urine samples were assayed for 2-OHE1 and 16alpha-OHE1 by using competitive enzyme immunoassay kits purchased from the ImmunaCare Corporation. The coefficients of variation for urinary 2-OHE1/16alpha-OHE1 over the study period ranged from 13.7 to 59.6% (mean, 33.3%) in our study participants. There was a good correlation between the level of the urinary 2-OHE1/16alpha-OHE1 ratio in any single urine sample and the average ratio over the 8-week study period from the same woman, with the mean correlation coefficient of 0.85. These results indicated that the within-person variation of the 2-OHE1 to 16alpha-OHE1 ratio for most women was moderate and the level of this ratio in a single urine sample, in general, reflects reasonably well the level of this biomarker over a 2-month period.

Objective: The objective of this study was to assess the longer term (up to 7 years) functional status and quality of life outcomes from lower extremity revascularization., Methods: This study was designed as a cross-sectional telephone survey and chart review at the University of Minnesota Hospital. The subjects were patients who underwent their first lower extremity revascularization procedure or a primary amputation for vascular disease between January 1, 1989, and January 31, 1995, who had granted consent or had died. The main outcome measures were ability to walk, SF-36 physical function, SF-12, subsequent amputation, and death., Results: The medical records for all 329 subjects were reviewed after the qualifying procedures for details of the primary procedure (62.6% arterial bypass graft, 36.8% angioplasty, 0.6% atherectomy), comorbidities (64% diabetics), severity of disease, and other vascular risk factors. All 166 patients who were living were surveyed by telephone between June and August 1996. At 7 years after the qualifying procedure, 73% of the patients who were alive still had the qualifying limb, although 63% of the patients had died. Overall, at the time of the follow-up examination (1 to 7.5 years after the qualifying procedure), 65% of the patients who were living were able to walk independently and 43% had little or no limitation in walking several blocks. In a multiple regression model, patients with diabetes and patients who were older were less likely to be able to walk at follow-up examination and had a worse functional status on the SF-36 and a lower physical health on the SF-12. Number of years since the procedure was not a predictor in any of the analyses., Conclusion: Although the long-term mortality rate is high in the population that undergoes lower limb revascularization, the survivors are likely to retain their limb over time and have good functional status.

This study examined the relationship between dentofacial appearance and judgments of facial attractiveness in a sample of 60 subjects between ages 65 and 75. Subjects completed questionnaires regarding their current dental appearance and level of satisfaction with their facial attractiveness. Subjects then viewed and ranked four sets of five photographs that had been digitally manipulated to simulate common dental defects. Regardless of background facial attractiveness or gender of stimulus photographs, a specific ranking order for dental defects emerged with rank-order correlations in the 90% range. Findings suggest that dental appearance affects judgments of facial attractiveness regardless of gender or background facial attractiveness.

Objectives: Clinical pathways have been implemented nationwide but little is understood about their effects on efficiency of care and patient outcomes. The present study examined the effects of both development and implementation of two renal transplant pathways., Methods: Cohorts of patients at a university hospital were compared before, during, and after the development and implementation of two renal transplant clinical pathways: isolated renal transplant from cadaveric donors (n = 170) or from living donors (n = 178). Clinical pathways for cadaveric and living related donor renal transplants were developed and implemented. Hospital length of stay and complications and infections after renal transplant were determined., Results: Mean length of hospital stay decreased after development and implementation of the cadaveric donor pathway (11.8 days after implementation versus 17.5 days before development). Cadaveric kidney recipients also had statistically fewer complications and infections after both guideline development and guideline implementation (57.1% before, 24.5% during, 18.5% after), but the greatest effect occurred during development. All of these findings persisted after control for demographic and comorbid factors. There were no changes in hospital stay, complications, or infections in the patients who received kidneys from living donors., Conclusions: The development and use of a clinical pathway for cadaveric donor renal transplant patients was associated with a significant decline in length of stay, complications, and infections, but much of the effect was seen during development rather than during implementation, and a closely related pathway for living related donor patients had no effect. Further understanding of what factors predict an effective pathway and what elements (ie, development or implementation) have an effect should be undertaken.

Objective: The literature suggests that Medicare health maintenance organization (HMO) patients may have poorer outcomes with formal home-health care than do fee-for-service (FFS) patients, but it is unclear whether this is related to case-mix or quality. Our objective was to compare the home-health care outcomes for HMO and FFS Medicare patients after hospitalization for stroke, COPD, CHF, hip replacement, or hip fracture with fixation or replacement while controlling for site of discharge and other patient characteristics., Design: Patients were identified before hospital discharge with data collected at that time and then prospectively for 1 year., Setting: Nineteen acute general hospitals in Minneapolis/St. Paul, Minnesota., Patients: All Medicare patients in the above hospitals identified predischarge with stroke, COPD, CHF, hip replacement, or hip fracture with fixation or replacement., Outcome: weighted ADL scale and hospital readmission. Independent factors: site of discharge, HMO status, comorbidity, severity, and demographic factors., Results: A total of 970 subjects were studied, 211 of whom were discharged to home-care. HMO patients were more likely to be discharged to a nursing home than to home-care after controlling for other factors (OR = 1.7; P = .015). After controlling for site of discharge and patient characteristics through either propensity scores or regression analysis, there was no statistically significant difference in ADL function at 6 weeks or at 6 months between HMO and FFS patients. Nor was there was a statistically significant difference in hospital readmission rates at 6 weeks and 6 months between HMO and FFS home-care patients., Conclusions: The outcomes of Medicare HMO patients discharged to home-care are not worse than those of FFS patients.

Objective: To develop and test quality of care process measures for three medical conditions of nursing home patients: fever, shortness of breath, and chest pain., Design: Flowsheets designed to capture the critical elements of care for the above conditions were developed by an expert panel. Nursing home residents charts were reviewed retrospectively using the flow sheets. The reviews were translated into clinical scenarios, and the quality of care the scenarios represented was rated by an expert panel., Setting: All nursing homes in Hennepin County, MN, that care for Medicaid patients., Patients: A random sample of 1405 Medicaid nursing home residents from 1984 and 1988., Measures: Measures of quality of physician assessment and intervention, quality of nurse assessment and intervention, and global quality were developed and the intra- and interrater reliability were tested. The measures' validity was assessed by their ability to predict resident death., Results: Intrarater reliability was measured as the correlation of the ratings of blinded duplicates. The correlation for the global scale and the four subscales ranged from .74 to .88 (P < .001 for all). Interrater reliability was tested by examining what percentage of the quality ratings were within one unit (1-5 scale) for all three raters. All three raters were within one unit for more than 72% scenarios for all scales. The subscale of quality of physician assessment was able to predict resident death when the worst episode of care (OR = .47, 95% CI(.31-.74)) or the mean episode of care (OR .54, 95% CI(.30-.99)) was used. None of the other subscales or the global measure predicted death., Conclusions: Through the use of an expert panel, measures of nursing home quality of care were developed for shortness of breath, fever, and chest pain. These measures have reasonable reliability and significant face validity. Their validity is supported further by the ability of one of the measures to predict resident death.

We have previously found that for acetaminophen kinetic differences exist between the hepatic microsomal catalyzed protein binding and cysteine conjugation. We have also observed that the protein binding of acetaminophen is only to intralumenal proteins. Together these data suggested that two pools of the reactive metabolite, N-acetyl-p-benzoquinone imine (NABQI), are formed during the oxidative metabolism of acetaminophen: one on the cytosolic surface and the other within the lumen of the microsomes. This would indicate that some of forms of cytochrome P450 (CYP) catalyzing NABQI formation have their active site on the cytosolic surface and others on the lumenal surface. We have examined this question by comparing the rates of cysteine conjugation and protein binding of acetaminophen by microsomes from lymphoblasts transfected with the cDNAs for human CYPs. We found that CYP2D6 catalyzed only cysteine conjugation; CYP1A2 and 3A4 catalyzed only protein binding; CYP2E1 catalyzed both; and CYP1A1, CYP2A6 and CYP2B6 catalyzed neither. These data suggest that CYP2D6 has its active site only on the cytosolic surface; CYP1A2 and CYP3A4 only on the lumenal surface; and CYP2E1 has catalytic sites on both the lumenal and cytosolic surfaces of the membrane. In mouse studies we have found that ethanol administration increased acetaminophen protein binding by 265% but cysteine conjugation by only 61%. CYP2E1 and CYP2B increased, whereas CYP3A decreased and the others did not change. These data suggest that in control mice CYP2E1 catalyzes the bulk of protein binding, whereas CYP2D catalyzes slightly more cysteine conjugation than does CYP2E1.

The apparent association in the published literature of gender and age with dental fear and anxiety is far from consistent or universal. A random, age-stratified telephone survey of 398 adults was performed in a US metropolitan area: Denver, Colorado. Information collected included Kleinknecht's Dental Fear Survey (DFS). In addition to total DFS scores, values were also calculated for the sums of the five DFS physiologic response items (PATRESP) and 12 DFS fear-producing stimulus items (DENTSTIM). Tests for reliability of these three scales were performed (alpha = 0.804 to 0.936). In this sample population, significant age and gender differences were noted. In general, fear and anxiety decreased in importance with increased respondent age, with the largest difference noted between the 40-50 and 60-69 age groups. Increased fear and anxiety were most apparent among younger females (20-30 and 40-50) as compared with older females (60 and older). However, among males, the summary variable for physiologic response to fear and anxiety did not appear to be age-related. Among the oldest respondents (ages 70+), 12.2% did report a "major" response to muscle tension when in the dental chair. Females reported more fear of some stimuli associated with dental care (e.g., "feeling the drill in the mouth") than did males.

A large body of evidence indicates that acetaminophen toxicity is mediated through the formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NABQI). Two assays have been employed to monitor NABQI formation by hepatic microsomes: the conjugation with a thiol trap, such as cysteine or glutathione, and the binding of NABQI to microsomal proteins. Studies from our laboratory with rat hepatic microsomes have suggested that the two assays may not be equivalent. We now find with mouse hepatic microsomes that there are also marked differences between these two assays. Among these the rate of cysteine conjugation was almost three orders of greater than that of protein binding. Furthermore, ethanol feeding increased protein binding by 97%, but cysteine conjugation by only 33%. Protein binding was linear for 20 min while cysteine conjugation was linear for only 5 min. CO, imidazole and metyrapone inhibited cysteine conjugation much more than protein binding while SKF-525A and KCN had similar effects on both reactions. Both reactions increased linearly with increasing [NADPH] up to 0.32 mM. At higher concentrations, the rate of cysteine conjugation markedly decreased while the rate of protein binding plateaued. The addition of equimolar concentrations of NADH decreased protein binding, but had no effect on cysteine conjugation. NADPH reduced the protein binding of added NABQI while NADH had little effect. The reduction of NABQI back to acetaminophen was equal for NADPH and NADH. These data indicate that the formation of the cysteine conjugate of NABQI has markedly different kinetic characteristics than the microsomal protein binding. These data might suggest that the two reactions are catalyzed in part by different isoforms of cytochrome P-450.

Numerous in vitro studies have indicated that acetaminophen is activated by mouse hepatic microsomal cytochrome P450 to form N-acetylbenzoquinone imine. This in turn covalently binds through a Michael addition to protein sulfhydryl and amino groups. Although acetaminophen adducts of several cytosolic proteins have been purified after its administration in vivo, no adducts of specific microsomal proteins have been reported. We find that, after the in vitro incubation of mouse hepatic microsomes with [ring-14C] acetaminophen in the presence of an NADPH generating system, 95% of the bound radioactivity was associated with adducts to three intraluminal microsomal proteins: calreticulin and the two forms of thiol:protein disulfide oxidoreductase, Q2 and Q5. The acetaminophen bound to 0.35, 1.32, and 0.25 mol/mol of the three proteins, respectively. Sequencing of the 14C-labeled tryptic peptides indicated that the acetaminophen bound to lysine 103 of Q2, lysines 202, 209 or 210 and 354 of Q5 and lysines 233 or 239 of calreticulin. No adducts of cysteine residues were observed. Our data might suggest that acetaminophen hepatotoxicity results from the formation of the reactive metabolite within the endoplasmic reticulum. This then binds to these essential proteins and blocks the posttranslational modification of secretory and membrane proteins. This inhibition could then lead to cellular injury and death.

Gingival hyperplasia is a well-known complication of therapy with cyclosporine, calcium channel blockers, and phenytoin. It is characterized by the presence of inflammation and a marked fibrotic response. The mechanism of this adverse reaction is unknown. We propose that it may be initiated by the metabolic activation of these drugs to form reactive metabolites. These then cause cellular injury and lead to the gingival hyperplasia. To evaluate this hypothesis we examined phenytoin metabolism and the cytochrome P450 contents of gingival tissues from 10 patients undergoing surgery for various periodontal conditions. We found that microsomes obtained from the gingiva show significant phenytoin hydroxylase activity as determined by the production of 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) (range, 12.8 pmol HPPH/min.mg microsomal protein to 276.9 pmol HPPH/min.mg microsomal protein; rat control, 133.7 +/- 11.5 pmol HPPH/min.mg microsomal protein). We also found that CYP1A1, CYP1A2, CYP2C9, CYP2E1, and CYP3A4 were present in these microsomes. We detected no CYP2B6 or CYP2D6. We believe that these data support our hypothesis that the proliferative inflammation observed with drugs such as phenytoin, nifedipine, and cyclosporine may be initiated by the formation of reactive metabolites and that the formation of these metabolites may be catalyzed by one or more CYPs found in the gingiva. These metabolites may then cause cellular injury and induce a reactive inflammatory response, followed by fibroblastic proliferation. This proliferation leads to the excess collagen deposition observed with gingival hyperplasia.

We have extensively purified three of the hepatic microsomal intralumenal Ca2+-binding proteins, CBP1, CBP2, and CBP3, which were originally described by Van et al. [(1989) J. Biol. Chem. 264, 17494-17501]. These apparently homogeneous preparations showed only single 45Ca2+ binding bands. On the basis of the peptide sequence, CBP2 was found to be highly homologous with the previously described protein ERp72. Similarly, CBP3 was identical to calreticulin and CBP1 had some homology to calmodulin. Contrary to the report of Van et al. (1989), we found that CBP2 had little thiol:protein disulfide oxidoreductase activity. Of the three purified preparations, only CBP2 exhibited apparent intrinsic protein kinase activity. This activity was found to be due to contamination of the CBP2 preparation by an extremely low concentration of tightly bound casein kinase 2 (CK2). In line with this observation, the phosphorylation was inhibited by heparin, removed by antibody to CK2, and stimulated by spermine. Furthermore, CBP2 was readily phosphorylated in vitro by added CK2 but only slowly phosphorylated by several other protein kinases. Thus, the persistence of CK2 in a highly purified preparation of CBP2 along with several other lines of evidence presented in this study might suggest that the protein CBP2 is a physiologically relevant substrate for CK2. Furthermore, these data suggest that CK2 might be localized in the lumen of the endoplasmic reticulum and that the phosphorylation of CBP2 in the lumen may play a role in the chaperone activity attributed to this protein.

A screening program for cystic fibrosis (CF) heterozygotes was conducted in a large HMO prenatal population, to evaluate the level of interest among eligible patients, the effectiveness of prescreening education, attitudes toward the screening process, psychological effects, and utilization of prenatal diagnosis and its outcomes. The heterozygote identification rate and frequency of specific CFTR mutations were also assessed. Identified carriers were offered genetic counseling and testing of male partners. Prenatal diagnosis was offered if both parents were identified as carriers. A total of 5,161 women underwent carrier testing; 947 others completed survey instruments only. The acceptance rate of screening was high (78%), and pretest education by videotape was generally effective. Adverse psychological effects were not reported. Participants generally found screening to be desirable and useful. Screening identified 142 female heterozygotes, 109 couples in which the male partner was not a carrier, and 7 high-risk couples. The incidence of R117H mutations was much higher than expected. The number of identified carriers was much lower in Hispanics than in Caucasians. We conclude that large-scale prenatal screening for CF heterozygotes in the absence of a family history of CF is an acceptable method for identifying couples at risk for affected fetuses. Sufficient pretest education can be accomplished efficiently, test insensitivity is well accepted, adverse psychological events are not observed, and general patient satisfaction is high.

Objective: To examine the roles played by changes in case-mix, quality of care, and aggressiveness of care in explaining the 42% increase in mortality of the Medicaid nursing home population of Hennepin County, Minnesota, between 1984 and 1988., Design: Retrospective chart review., Setting: All nursing homes in Hennepin County, MN, that care for Medicaid patients., Patients: A random sample of 1605 Medicaid nursing home residents from 1984 and 1988 stratified by year and by whether the resident died in that year. Sampling was disproportionate to allow approximately 400 individuals per stratum. A total of 1405 charts (87%) were reviewed; the remainder were either lost or destroyed., Main Outcome Measures: Measures included case-mix (Charlson index, functional status, implicit reviewer assigned severity [range 1-4]), aggressiveness of care (orders limiting care), quality of care (process of care for tracer conditions [range 1-5], falls), and resident death., Results: Implicitly rated severity of illness worsened between 1984 and 1988 (2.77 vs 2.91; P = .009), but other measures of case-mix were unchanged. A greater percentage of residents had a DNR order in 1988 (12% in 1984 vs 37% in 1988; P < .001), and more received less aggressive care (31% vs 40%; P = .006). Overall process of care improved between 1984 and 1988 (2.88 vs 3.01; P < .05). With adjustment of the mortality rates and with logistic regression controlling for age and gender, changes in quality of care alone accounted for less than 5% of the mortality rate change between 1984 and 1988, case-mix alone accounted for 49%, and aggressiveness of care alone accounted for nearly 100%., Conclusions: The nursing home population became sicker between 1984 and 1988, but process of care improved. These changes had a modest effect on the mortality rate. The increase in less aggressive care between 1984 and 1988 accounts for nearly all of the increase in mortality.

The insecticide methoxychlor is estrogenic in birds and mammals and interferes with sexual development and reproduction, but it is not known whether this toxicity is due solely to its estrogenicity. We now have found that during hepatic, microsomal metabolism of [ring-14C]- or [3H-OCH3]methoxychlor, their metabolite primarily binds to iodothyronine 5'-monodeiodinase, type I (5'-ID1). The purified, radiolabeled protein reacted with antibodies against protein disulfide isomerase, isoform Q5, which is highly homologous to 5'-ID1. Sequencing of the radiolabeled tryptic peptide indicated that methoxychlor bound to cysteine 372 or 375 or to lysine 376 of 5'-ID1. Treatment of rats with methoxychlor for 4 days decreased hepatic, microsomal 5'-ID1 activity from 2.94 to 2.20 nmol/min-mg prot (P < 0.02). Since 5'-ID1 catalyzes thyroxine conversion to the biologically active triiodothyronine, these data suggest that methoxychlor may interfere with thyroid hormone metabolism. This may be an additional factor in its environmental toxicity.

We have examined the pharmacokinetic interaction between isradipine and lovastatin in six male and six female, healthy, normotensive, human subjects after a single dose and after treatment for 5 days. The isradipine plasma concentrations were determined by a radioimmunoassay and the lovastatin serum concentrations by gas chromatography-mass spectrometry (GC/MS) and by the inhibition of the 3-hydroxy-3-methylglutaric-coenzyme reductase activity. We found that the apparent serum concentrations of lovastatin were 4- to 6-fold higher in the reductase-inhibition assay than the GC/MS assay, suggesting that the bulk of the reductase inhibition is due to active metabolites. The peak and the time-to-peak concentrations were unaffected by the treatments, either after the first dose or after continued administration. In male subjects, after repeated doses of isradipine, the lovastatin area under the time-concentration curves (AUCs) decreased by 40% as determined by the GC/MS assay (P < .001) and 20% as determined by the reductase-inhibition assay (P < .0022). In the female subjects, isradipine treatment decreased the lovastatin AUCs as determined by the GC/MS assay, but this was not statistically significant due to a high variance. Furthermore, in the female subjects, isradipine had no effect on the lovastatin AUCs as determined by the reductase-inhibition assay. Because the lovastatin peak and the time-to-peak concentrations were unaffected by isradipine treatment, the decreased lovastatin AUCs were probably not due to altered intestinal absorption. More likely, because lovastatin has a high hepatic clearance, the decreased AUCs seen after isradipine treatment could be due to increases in the clearance of lovastatin secondary to increased hepatic blood flow.

This is the first report of long-term use (one year) of isradipine, a new dihydropyridine calcium channel blocker, in the treatment of elderly patients with essential hypertension. Patients completing a three month, double-blind, multicentre study comparing isradipine to hydrochlorothiazide (HCTZ) were eligible to enroll in this open-label, continuation study. At initial baseline, patients were at least 60 years of age and had DBP from 95 mmHg to 120 mmHg. Patients were titrated when necessary every two weeks with isradipine, 5 mg to 15 mg once daily or 2.5 mg to 10 mg twice daily, to maintain sitting DBP < or = 90 mmHg. HCTZ, 12.5 mg to 50 mg once daily, could be added for better BP control. A total of 136 patients completed the one year, open-label phase. One hundred and fourteen patients (84%) received isradipine as monotherapy (mean dose, 9.7 mg/day); 22 received concomitant HCTZ therapy at one year. Reduction in DBP was significant and similar among all age groups and races (mean change of -19 mmHg). Reduction in SBP was similar among all age groups. Ninety-four per cent of those receiving isradipine monotherapy achieved BP control during the last four months of treatment. Twenty-six patients (16%) withdrew from the study: 11 (7%) had adverse reactions (one with headache, two with pedal oedema, eight with other problems); 11 (7%) had nondrug-related problems; and in four (2%), the drugs were ineffective. Based on these observations, isradipine is a well-tolerated, safe and effective agent for long-term BP control in elderly patients with essential hypertension.

Cigarette smoking is a leading risk factor for atherosclerosis. Endothelial injury may be the initial event in this process. The carcinogenic metabolites of the polycyclic aromatic hydrocarbons found in cigarette smoke tars could cause this injury. We tested this model by examining the effect of 3-methylcholanthrene administration on aortic polycyclic aromatic hydrocarbon metabolism. Immunoblotting with a monoclonal antibody (mAb 1-7-1) specific for cytochromes CYPIA1 and CYPIA2 showed that aortic microsomes from treated, but not from control, animals contained CYPIA1; the CYPIA1 was primarily in the endothelium. Aortic microsomes from induced animals metabolized benzo[a]pyrene (BaP) to the 7R,8S,9,10-tetrahydrotetrol-, 7,8-dihydrodiol-, 1,6 quinone-, 3,6 quinone-, 6,12 quinone-, 3-hydroxy-, and 9-hydroxy-BaP. mAb 1-7-1 inhibited the formation of the tetrahydrotetrol, the dihydrodiol-BaP, and the 3-hydroxy-BaP but did not inhibit the quinones or the 9-hydroxy-BaP. Arachidonic acid did not affect metabolism. These data suggest that the aortas of induced animals metabolize the BaP in cigarette smoke to carcinogenic and toxic products and that this metabolism may initiate vessel injury and lead to the accelerated atherosclerosis seen in cigarette smokers.

Objective: To examine changes in the aggressiveness of care of the nursing home population and to determine the factors that predict whether care is limited., Design: Retrospective chart review., Setting: All nursing homes in Hennepin County, Minnesota that care for Medicaid patients., Patients: A random sample of 1605 residents of Hennepin County, MN nursing homes from 1984 and 1988 who were also Medicaid beneficiaries. The sample was stratified by year and by whether the resident died in that year. The residents were sampled disproportionately to allow approximately 400 individuals in each stratum. A total of 1405 charts (87%) was reviewed; the remainder had either been lost or destroyed., Main Outcome Measure: Orders limiting care (do not resuscitate, supportive care only, etc.), care that was less aggressive than care usually given to a person of that age (eg, not using antibiotics for a clear infection or not sending a resident to the hospital for an illness for which someone would "usually" be sent to the hospital), determined implicitly through chart review., Results: The demographic characteristics of the nursing home population did not change between 1984 and 1988, but the population had significantly greater severity of illness in 1988. The percentage of residents with 'do not resuscitate' orders (DNR) increased from 12% to 37% (P < 0.0001), and the use of other orders to limit care (do not hospitalize, supportive care only, etc.) increased from 12% to 17% (P < 0.05). The use of CPR did not change between the two years but the percentage of residents who were found to receive less aggressive care through implicit chart review increased from 31% to 40% (P < 0.01). The year the resident was in the nursing home, the severity of illness, functional status, and dementia were significant predictors of DNR status. Factors that predict receipt of less aggressive care were similar except that age was also a significant predictor., Conclusions: The nursing home population received less aggressive care in 1988 than in 1984. Further, there was a discrepancy between what was ordered and what was delivered in the nursing home in that residents with DNR orders had care limited beyond the withholding of CPR.

We have previously shown that the cDNA sequence published for the rat gene encoding phosphatidylinositol specific phospholipase C alpha (1) may be identical to the sequence of a new isoform of protein disulfide isomerase, Isozyme Q2 (2). To determine whether the presumed phospholipase C alpha gene encodes phospholipase C or protein disulfide isomerase, we have now expressed the cDNA in Escherichia coli and find that the 55.5 kDa protein that it synthesized contains no phospholipase C activity. Moreover, an extract of Escherichia coli containing the expressed protein shows a concentration dependent increase in protein disulfide isomerase activity. The expressed protein is identical to protein disulfide isomerase, Isozyme Q2 on SDS-PAGE and by Western blot analysis. These current studies confirm that the published sequence of phospholipase C alpha is, in fact, the sequence of protein disulfide isomerase, Isozyme Q2.

The aim of this study was to assess the attitudes of dental students towards the elderly in the two dental schools in Israel. The study population comprised 180 dental students currently enrolled in the two dental schools in Israel. The response rate was 59% and 92% for the students in Tel Aviv and Hebrew University respectively. The self-administered questionnaire measured attitudes using the Rosencranz and McNevin Aging Semantic Differential Scales. Students' attitudes were observed to be more or less neutral. No differences of significance were observed between the students of the two schools. Social contact with and experience in treating the elderly did not seem to change students' attitudes and their desire to work with elderly patients in the future. Further development of the curriculum should be implemented in order to try to change students' attitudes towards the elderly from neutral to positive for the benefit of the provider and society.

The effects of selenium, vitamin E, and DL-methionine deficiency on the acute lethality and hepatotoxicity of acetaminophen in male CD-1 mice were studied. Vitamin E and selenium deficiencies led to an increase in the acute lethality of acetaminophen, with a decrease in the LD50 from 376 to 84 mg/kg. These dietary deficiencies impaired the inducibility of the hepatic microsomal mixed function oxidase system by phenobarbital, but on the basis of the covalent binding of acetaminophen to microsomes, these treatments did not alter the activation of acetaminophen to a reactive intermediate by this system. Addition of methionine to the deficient diet restored hepatic glutathione content to control levels but did little to protect against the acute lethality of acetaminophen. In methionine-supplemented animals, the addition of either selenium or vitamin E increased the LD50 of acetaminophen to 167 and 200 mg/kg, respectively. Administration of a sublethal, toxic dose of acetaminophen (LD30) to the methionine-supplemented and selenium- and vitamin E-deficient mice did not produce any hepatic damage as evidenced by a lack of plasma aminotransferase elevation. In view of the known antioxidant effects of vitamin E and selenium, these data suggest the involvement of a reactive radical in the acute lethality of acetaminophen and further suggest that death from acute acetaminophen overdose in chronic selenium- and vitamin E-deficient mice may be unrelated to liver necrosis.

Thiol:protein-disulfide oxidoreductase catalyzes the GSH reduction of protein disulfides to sulfhydryls. Chromatography of solubilized hepatic microsomes on Mono Q yielded two peaks, Q-2 and Q-5, which contained all the thiol:protein-disulfide oxidoreductase activity. These were further purified by chromatofocusing giving specific activities of 14.4 and 45.9 nmol/mg of protein/min, respectively with purifications of 45.0- and 143.6-fold. Amino acids 1-18 of Q-5 were the same as previously reported for Thiol:protein-disulfide oxidoreductase (Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317, 267-270), except amino acid 1 was leucine instead of aspartate and amino acid 6 was asparagine instead of glutamate. The N-terminal amino acid sequence of Q-2 differed markedly from Q-5 but Q-2 showed 100% identity at amino acids 25-54, 258-269, 285-310, 347-350, 412-419, and 434-463 for the reported sequence of rat, hepatic, cytosolic phosphatidylinositol-specific phospholipase C form 1a (PLC) (Bennett, C. F., Balcarek, J. M., Varrichio, A., and Crooke, S. T. (1988) Nature 334, 268-270). PLC activity was found in the elution from the Mono Q column, but none was found in purified Q-2 or Q-5. Antibodies to Q-5 reacted with Q-2, but anti-Q-2 did not react with Q-5. Anti-Q-2 antibody showed immunoreactivity with 55- and 60-kDa microsomal proteins, whereas Q-5 antibody reacted with a number of microsomal proteins. Although Q-2 was immunoreactive with a polyclonal antibody to guinea pig, uterine cytosolic PLC, partially purified PLCs from rat liver cytosol did not react to this antibody. Our data would suggest that the published sequence for PLC form 1a may actually be the sequence for Q-2.

Glaucoma screening is a standard procedure at many health fairs. Information on correct screening techniques, instrumentation, and target populations has been available. However, a scarcity of information exists concerning the success of efforts to follow up on abnormal results. This study reports on the findings of a long-term (6-month) follow-up of all individuals with abnormal tonometric results screened at an inner-city osteopathic hospital. Of the 218 subjects screened, 15 were found to have abnormal tonometric results. Seven of the 15 subjects were actually reached at the 6-month follow-up interval. Four of the seven had not sought any further eye care; two had sought nonmedical evaluation; only one had sought medical ocular care, and that subject was later found to have glaucoma. The results presented in this article indicate that compliance by this population is inadequate. Inner-city participants require more education. We encourage physicians to promptly refer such patients for appropriate medical ocular care.

This paper reports the results of a national survey of 1,321 general practitioners regarding their level of awareness of and experiences with the treatment of older victims of abuse and neglect. Greater than 90% were aware of elder abuse and neglect as a social and medical problem. Greater than 10% had personal knowledge or had treated a victim of abuse or neglect. Data suggest that dentists will increasingly come in contact with victims and must be trained to recognize, treat, and report such incidents.

In hepatic microsomes one or more isozymes of cytochrome P-450 inhibits the ATP-dependent Ca2+ pump. This inhibition is reversible by GSH and appears to be due to a direct oxidation of the pump proteins by the oxygenated cytochrome. To determine which isozyme mediates this inhibition, we have examined the effect of various substrates and inhibitors on the NADPH inhibition of Ca2+ uptake. We find that aminopyrine, benzphetamine and SKF-525A reverse this inhibition while a number of other substrates do not. This pattern suggests that a previously unreported isozyme of cytochrome P-450 mediates the Ca2+ pump inhibition.

After 8 weeks of isradipine, a twice-a-day dihydropyridine calcium channel blocker, 49% of elderly patients showed a complete response (sitting diastolic blood pressure less than or equal to 85 mm Hg) and 36% showed a partial response (sitting diastolic blood pressure decrease greater than or equal to 10 mm Hg) for an 85% total response rate. Hydrochlorothiazide gave a complete response in 36% of the patients and a partial response in 33%, for a 69% total response rate (p less than 0.0046). Because elderly subjects have reduced clearance for many drugs, we determined how those who responded to twice-a-day administration would respond to once-a-day administration. After 4 weeks of isradipine administered once a day, 54% of the patients showed a complete or partial response, whereas 38% of the patients who were changed to placebo showed a response. In contrast, 82% of patients receiving hydrochlorothiazide once a day showed a response, whereas 60% of patients who were changed to placebo showed a response. These data indicate that the standard formulation of isradipine was not effective when administered once a day.

Acetaminophen is metabolized by cytochrome P450 to N-acetyl-p-benzoquinone imine (NABQI). This metabolite reacts with critical cellular macromolecules to give toxicity. The administration of 10% ethanol in the drinking water to 100 g male rats for 6 weeks markedly increases the toxicity of acetaminophen. This increase was associated with a 71% increase in microsomal protein binding of acetaminophen [4.8 pmol/min/mg protein in control microsomes versus 8.2 pmol/min/mg protein in ethanol microsomes (P less than 0.01)] and a 131% increase in aniline hydroxylase [0.52 nmol/min/mg protein in control microsomes versus 1.20 nmol/min/mg protein in ethanol microsomes (P less than 0.001)]. On the other hand, cysteine conjugation of acetaminophen showed an increase of only 12% [2.8 nmol/min/mg protein in control microsomes versus 3.1 nmol/min/mg protein in ethanol microsomes (P less than 0.05)]. Ethylmorphine- and benzphetamine N-demethylases did not increase. In microsomes from both control and ethanol animals, imidazole (1 mM) inhibited the two N-demethylases, aniline hydroxylation and acetaminophen binding by 85-95% but inhibited the cysteine conjugation by only 50%. For control and ethanol animals, both 80% CO/20% O2 and SKF-525A (1 mM) totally inhibited cysteine conjugation but only inhibited the other activities by about 36-60%. KCN (1 mM) had no effect on any of the activities except protein binding (60-67% inhibition). Scavengers of reactive oxygen [mannitol (1 mM), dimethyl sulfoxide (1 mM), superoxide dismutase (15 micrograms/mL) and catalase (65 micrograms/mL)] had no effect on any of the reactions. Of all these treatments only CO/O2 decreased the protein binding and cysteine conjugation of NABQI in the presence of either NADP+ or NADPH. The data from the inhibitor studies and the effect of ethanol on acetaminophen and NABQI metabolism would suggest that protein binding and cysteine conjugation are catalyzed by different isozymes of cytochrome P450. Finally, the current results indicate that the increased toxicity of acetaminophen observed with ethanol more closely parallels the increase in protein binding activity rather than cysteine conjugation.

This paper reports the results of a study of dentists' recommendations regarding the appropriate health action of elders in response to certain oral common symptoms and problems. Previous studies have found that elders most commonly respond to oral symptoms by using nonprofessional means. This study found that, without exception, the recommendation to consult a dentist was the primary mode of response favored by dentists, even in response to symptoms and problems that are most often trivial and rarely indicative of serious health problems. The results of this pilot study suggest that the need exists for dentists to reach a more realistic consensus regarding alternatives to professional consultation in response to some symptoms and problems.

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

This article reports the results of a study of factors that differentiate among utilizers and nonutilizers of dental services in old age. Two hundred community-dwelling subjects aged 60-89 were surveyed regarding utilization of dental services, dentate status, current treatment needs, recent symptom experiences, and dental fear and anxiety. Discriminant analysis was used to differentiate among utilizers and nonutilizers. Dentate status, perceived need, and recent symptom experiences were the best predictors of utilization. The contribution to accurate classification made by two measures of dental fear and anxiety was marginal at best. When dentate status was removed as a predictor, the role of fear and anxiety became somewhat more important.

A number of experiments were conducted to determine to what extent retinal image smearing during saccades provides information about the eye movement magnitude to the perceptual system. The technique involved obtaining measures of perceived movement when the total visual field was displaced in conjunction with saccadic eye movements. Trials with normal retinal smear were compared with trials on which smearing was greatly reduced or eliminated. The results are interpreted as showing that the absence of normal retinal smear during a saccade increases the uncertainty in the information available to the perceptual system and that this uncertainty results in a tendency to perceive smaller than veridical amounts of movement.

Two improvements in propranolol resolution were developed. Both the (+)- and (-)-di-(p-toluoyl)tartaric acids were used as the resolving agents. This procedure reduced the number of crystallizations needed to obtain a pure product. Furthermore, synthesis of the resolving agent was improved.

Hepatic microsomes from male rats hydroxylate R-(+)- and (-)-[2-14-C]pentobarbital to the diasterioisomeric 3'-alcohols: metabolite I (1'RS, 3'SR)- and metabolite II (1'RS, 3'RS)-5-ethyl-5-(3'-hydroxy-1'methylbutyl) barbituric acids. The rate is linear up to 1.5 mg of microsomal protein per ml of incubation mixture and for up to 16 min. The metabolism of R-(+)-pentobarbital led to production of nearly equal quantities of metabolites I and II with a total hydroxylation of 1.08 nmol/min/mg of protein. On the other hand, the S-(-) pentobarbital gave 3- to 5-fold less of metabolite I than II and a total hydroxylation of 1.42 nmol/min/mg of protein. The KM values for formation of metabolite I were 381 muM from R-(+)- and 241 muM from the S-(-)-pentobarbital, while for metabolite II they were 115 muM from R-(+)- and 68 muM from S-(-)-pentobarbital. These data suggest that there are at least two enzymes catalyzing the hydroxylation of pentobarbital, one to give metabolite II and another or others to give metabolite I.

Previous in vivo and in vitro studies have indicated that the Km for phenytoin hydroxylation is about 30 microM. Yet, the drug shows dose-dependent kinetics suggesting a Km of about 5 microM. The present studies indicate the discrepancy is not due to active transport of the drug in the hepatocyte or a decrease in the Km due to the low pO2 of the portal vein leading to uncompetitive inhibition. Studies in both hepatocytes and microsomes indicate the presence of a high affinity hydroxylase with a Km of 2 to 5 microM. These data suggest that this enzyme is the one primarily involved in the metabolism of phenytoin.

Displacement of spin-labeled drugs from drug-specific antibodies in human serum can be measured by use of electron spin resonance. This "spin immunoassay" has advantages of speed, simplicity, and small volume requirements over other standard techniques such as gas-liquid, liquid-liquid, or thin-layer chromatography. Unlike radioimmunoassay, there is no need to separate free and bound label and there are no radiation hazards. We present clinical and experimental data showing how this method can be applied to the routine determination of morphine and diphenylhydantoin concentrations in serum.