Sign of lipid peroxidation as measured in the urine of patients with probable Alzheimer's disease.

Free radical-induced oxidative damage may be involved in the neurodegenerative process associated with Alzheimer's disease (AD). 8-Isoprostaglandin F(2alpha) (iPF(2alpha)-III) is an isoprostane derived from free radical-induced non-enzymatic oxidation of arachidonic acid. It is formed in vivo and is an indicator of lipid peroxidation. Measurements were made of iPF(2alpha)-III in the urine of patients with mild to moderate dementia associated with probable AD and compared to those in the urine of non-demented subjects, who were similar in age and gender. 2,3-Dinor thromboxane B(2) (dinor TXB(2)), a urinary metabolite of TXB(2) was also measured, and served as an indicator of the enzymatic transformation of a product of arachidonic acid. Enzyme linked immunoassays were used to measure iPF(2alpha)-III and dinor TXB(2) in the urine. The concentration of iPF(2alpha)-III was significantly elevated in urine of patients assessed to have mild to moderate dementia as compared to non-demented patients. The concentration of urinary dinor TXB(2) was also significantly elevated in the patients with dementia and probable AD as compared to the non-demented subjects. There was considerable overlap of values obtained for demented and non-demented patients for iPF(2alpha)-III and dinor TXB(2), respectively. The observed elevation of iPF(2alpha)-III suggests that patients with mild to moderate dementia associated with probable AD are experiencing significant oxidative stress. This finding is consistent with current data suggesting that oxidative stress may be occurring in patients with dementia and probable AD. The increase of dinor TXB(2) may indicate that enzymatic processes related to the metabolism of arachidonic acid-derived products are also increased in demented patients with probable AD.

Free radical oxidative damage and Alzheimer's disease.

There is increasing evidence that free radical-induced oxidative damage may play a role in the pathogenesis of Alzheimer's disease. Free radicals are reactive oxygen compounds that may attack and damage lipids, proteins, and DNA. The brain is especially sensitive to oxidative damage because of its high content of readily oxidized fatty acids, high use of oxygen, and low levels of antioxidants. Evidence for oxidative damage has been obtained from postmortem brain tissue as well as from living patients with Alzheimer's disease. Antioxidants such as vitamin E show promise that they may help in treating the disease.

Opinions and reactions of physicians in New Jersey regarding the Oregon Death with Dignity Act.

Physician-assisted suicide (PAS) was legalized in Oregon in 1997. In the study reported here, the authors surveyed a sample of New Jersey physicians with regard to Oregon's Death with Dignity Act and to whether similar legislation should be enacted in New Jersey. A 49-item questionnaire was sent to 563 physicians in New Jersey who were licensed in the specialties of family practice, internal medicine, surgery, psychiatry, and obstetrics/gynecology. The questionnaire contained sections pertaining to demographics, physicians' attitudes regarding PAS, and physicians' opinions on Oregon's Death with Dignity Act. A brief summary of the legislation was included in the mailing, which participants were asked to read before completing the questionnaire. Of the 191 physicians who responded to the survey, 55% agreed with legislation that would legalize PAS, and 59% said that a law similar to that enacted in Oregon should exist in New Jersey. However, only 47% of respondents indicated that they believed PAS to be consistent with the role of a physician to relieve pain and suffering. Slightly more than half of respondents indicated that they would refuse to participate in PAS and were concerned about issues such as professional and personal liability and the potential for abuse. Physicians in New Jersey will require additional information, education, and discussion of the ethical and legal implications of PAS before a law similar to that in Oregon could be proposed or considered.

NMDA receptor antagonism produces antinociception which is partially mediated by brain opioids and dopamine.

Inhibition of nitric oxide synthase (NOS) activity results in opioid-mediated supraspinal analgesia in the rat, as indicated by increased reaction time in the hot plate test. It is documented that a relationship exists between NMDA receptor activation and the activity of NOS. The present investigation sought to determine if inactivation of the NMDA receptor produced antinociception of supraspinal origin, as was observed in response to inhibition of NOS, and if this response was mediated by brain opioids, by activation of receptors for the neurotransmitter, dopamine, or both. Administration of MK-801, a non-competitive antagonist of the NMDA receptor, produced significant antinociception as measured by reaction time in the hot plate test of analgesia. Antinociception resulting from treatment with MK-801 appeared to be mediated by brain opioids, as indicated by the ability of the opioid antagonist, naloxone, to partially reverse the effect of MK-801 administration. This analgesic response was also partially diminished by administration of the dopamine D1 receptor antagonist, SCH 23390 and the dopamine D2 receptor antagonist, sulpiride. The analgesia resulting from NMDA receptor antagonism was found to be only partially attributable to dopamine and brain opioids, since co-administration of naloxone and SCH 23390 or naloxone and sulpiride, were unable to completely reverse the antinociceptive response to MK-801. The present findings suggest that inhibition of NMDA receptor activity produces supraspinal analgesia. Furthermore, it appears that antinociception induced by blockade of the NMDA receptor results, at least in part, from activation of endogenous brain opioids and stimulation of D1 and D2 subtypes of the dopamine receptor.

Enzyme immunoassays (EIAs) of eicosanoids.

Enzyme immunoassay of eicosanoids is a nonradioactive, highly sensitive method of determining the concentration of eicosanoids in biological samples. Although relatively easy to use, the assays require a high level of precise pipetting technique and familiarity with critical points in the assay procedure. Although assay kits complete with plates, buffers, antibodies, tracers, and color development reagents are available, it is more economical to develop the assay within the laboratory if the assay is to be performed routinely. The only major disadvantage with EIA is that the investigator is limited to measuring eicosanoids with commercially available enzyme-tracers and antibodies. The labeling of particular eicosanoids by enzyme tracers is rarely, if ever, performed outside of industry. Growing of antibodies is conducted in many laboratories but is beyond the scope of this chapter. It requires a significant level of commitment of time and resources to establish the specificity of the antibody (i.e., does the antibody cross-react with eicosanoids of similar structure). Furthermore, this will not solve the problem of availability of eicosanoid-tracer. On the other hand, it must be noted that with the exception of the cytochrome P-450 metabolites of arachidonic acid, most of the major eicosanoids that are biologically active and are known to play regulatory roles in physiology and/or pathology, have commercially available antibodies and enzyme-tracers.

Augmentation of nitric oxide, superoxide, and peroxynitrite production during cerebral ischemia and reperfusion in the rat.

The effect of ischemia produced by bilateral occlusion of the common carotid arteries (30 min) followed by 4 hours of reperfusion on total and inducible nitric oxide synthase (NOS) activity and the production of nitric oxide (NO), superoxide and peroxynitrite in the cerebral hemispheres was determined in the rat. Compared to sham-operated controls, cerebral ischemia-reperfusion resulted in a significant increase in total and inducible NOS activity and a significant increase in the production of NO and superoxide in the cerebral hemispheres. The level of NO in the plasma and the peripheral leukocyte count were also significantly increased. Immunohistochemical staining for nitrotyrosine (a marker of peroxynitrite production) showed that ischemia-reperfusion resulted in increased synthesis of cerebral peroxynitrite. Administration of the irreversible NOS inhibitor, Nomega-nitro-L-arginine (L-NA), increased superoxide levels in the brain and significantly reduced plasma NO. Total and inducible NOS activity as well as NO and immunoreactive nitrotyrosine, in the cerebral hemispheres were reduced with L-NA administration. The number of leukocytes in the plasma was unaffected by administration of L-NA. These findings suggest that cerebral ischemia-reperfusion causes increased production of reactive oxygen species in the cerebral hemispheres and that the production of peroxynitrite, and not superoxide, may be dependent upon the availability of NO.

Cocaine and inhibition of nitric oxide synthesis produce opioid-mediated antinociception.

Cocaine and nitric oxide are known to influence the perception of pain. The present study sought to determine if the endogenous opioid peptide system participates in cocaine-induced antinociception and antinociception produced by antagonism of nitric oxide. Pain perception was measured using the hot plate test. Administration of cocaine (25 mg/kg) to male rats resulted in a significant increase in reaction time in the hot plate test, which was reversed by treatment with 3, 10, and 30 mg/kg of the opiate antagonist, naloxone. In rats that were not treated with cocaine, doses of 30 and 60 mg/kg of naloxone significantly reduced hot plate reaction Time. Treatment of animals with the nitric oxide synthase inhibitor, N omega nitro-L-arginine, produced a significant increase in response time to the hot plate, which was reversed by administration of naloxone. These data indicate that antinociception produced in the rat by cocaine appears to have a supraspinal component and to involve activation of endogenous opioid peptide activity in the brain. The results also suggest a tonic inhibition of endogenous opioid peptide activity by nitric oxide, which when antagonized, results in diminished response to pain.

Effect of transient overexpression of Gq alpha on soluble and polymerized tubulin pools in GH3 and AtT-20 cells.

In order to study Gq-tubulin interaction in the cytosol, GH3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with Gq alpha cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by Gq alpha-transfected GH3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that Gq alpha-specific staining was much more prominent in Gq alpha-transfected GH3 and AtT-20 cells (also transfected with Gq alpha) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional Gq alpha. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in Gq alpha-transfected GH3 and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in Gq alpha-transfected GH3 and AtT-20 cells. To determine whether these effects on tubulin were mediated by Gq directly, we examined the influence of purified Gq on tubulin polymerization. Gq (0.5 microM) inhibited polymerization of crude tubulin (present in GH3 cell cytosol) by 53%. In contrast to its effects on GH3 cell cytosol tubulin, Gq stimulated purified tubulin polymerization by 160%. These results suggest that Gq modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components.

Effect of colchicine and taxol on thyrotropin-releasing hormone receptor coupling to G protein in GH(3) cells.

The role of membrane-associated tubulin in TRH receptor-G protein coupling was investigated with the use of compounds that influence tubulin function. TRH-stimulated G protein GTPase activity in GH(3) cell membranes was used to determine receptor-G protein coupling. TRH-stimulated GTPase activity was abolished by G(qα) antibody, suggesting that TRH receptor coupling to G(q) results in the activation of G(qα) and the subsequent hydrolysis of GTP. TRH (1 µM) stimulated the enzymatic activity by up to 69 pmol/min/mg protein, and in the presence of 1 µM colchicine the hormone-stimulated activity was only 26 pmol/min/mg protein. Similar inhibition of TRH receptor-G protein coupling was observed with tubulin antibodies and purified tubulin, suggesting that perturbation of membrane-associated tubulin and/or tubulin-G protein interaction by these compounds disrupts receptor-G protein interaction. Next, the events occurring at the initial stages of TRH-mediated signal transduction were correlated to prolactin (PRL) secretion in GH(3) cells. Colchicine (1 µM) and taxol (1 µM) inhibited the basal PRL secretion by 38 and 44%, respectively. In addition, colchicine (1 µM) and taxol (1 µM) significantly inhibited TRH-stimulated PRL secretion. TRH-stimulated PRL secretion in control, colchicine-, and taxol-treated cells was 13.9, 9.1, and 6 ng/mL, respectively. Furthermore, polymerized tubulin levels were decreased by colchicine and increased by taxol. These results suggest that perturbation of the steady state of tubulin-G(q) interaction may disrupt the initial events in TRH-mediated signal transduction.

Occurrence and impact of suspected delirium in hospitalized elderly patients.

The records of 95 consecutive people older than 65 years and admitted to a community hospital were retrospectively reviewed to determine the prevalence of undiagnosed delirium in hospitalized elderly patients. Chart review focused on identification of patients with documented diagnosed delirium according to Diagnostic and Statistical Manual of Mental Disorders, third edition, revised (DSM-III-R) criteria and patients with unrecognized delirium. Unrecognized delirium was considered present when information contained in a patient's chart met some or all criteria for delirium as described in DSM-III-R, but the physician's progress notes failed to indicate delirium as a diagnosed clinical entity. The prevalence of diagnosed delirium was 2%. Thirty-six percent of the patients were suspected of having unrecognized delirium. The mean length of hospital stay and the rate of mortality were significantly higher for patients with suspected delirium than for non-delirious patients. The findings of this study suggest that unrecognized delirium in the hospitalized elderly may occur frequently and is associated with an increased length of hospital stay and increased mortality.

Effect of thyrotropin-releasing hormone on microtubules in GH3 cells.

The effect of thyrotropin-releasing hormone (TRH) on the tubulin-microtubule system in GH3 cells was investigated. Prolactin (PRL) secretion by GH3 cells was stimulated by TRH at 1, 5, and 10 min of incubation. The polymerized tubulin levels increased from 0.4 units micrograms-1 protein in control cells to 0.6 units micrograms-1 protein in cells incubated with TRH for 1 min. Soluble tubulin levels increased from 0.8 units microgram-1 protein in control cells to 1.2 units micrograms-1 protein in cells exposed to TRH for 5 min. Tubulin levels were similar in control cells and cells treated with TRH for 10 min. These results suggest that TRH-stimulated PRL secretion is accompanied by a shift in the equilibrium between tubulin pools.

Comparison of the effects of immobilization and pressure overload induced cardiac hypertrophy on immunoreactive beta-endorphin.

Acute physical stress in the form of immobilization resulted in a decrease in the concentration of immunoreactive beta-endorphin (IR-BE) in the anterior pituitary (AP) and an increase in the concentration of IR-BE in the neurointermediate lobe of the pituitary (NIL) and the plasma. Hypothalamic IR-BE was not influenced by immobilization. In response to chronic cardiovascular (physiological) stress resulting from constriction of the aorta (aortic banding) and subsequent pressure overload, the concentration of IR-BE in the AP was increased as was the concentration of IR-BE in the plasma. The concentration of IR-BE in the NIL and the hypothalamus was not affected. These findings suggest that physical stress and cardiovascular stress have the same affect on IR-BE levels in the plasma but differ in their respective effects on IR-BE in the AP and NIL and do not affect the concentration of IR-BE in the hypothalamus. The difference in the effects of each form of stress on the AP and the NIL respectively, may be attributed to either the type of stress employed (physical versus physiological), the duration of the stress (acute vs chronic), or both.

Vinblastine and nocodazole inhibit basal and thyrotropin-releasing hormone-stimulated prolactin secretion in GH(3) cells.

To investigate the efficacy of vinblastine as a possible therapeutic agent in prolactinomas, we have examined the effects of vinblastine on GH(3) cell function. The effects of vinblastine were compared to another anti-microtubule drug, nocodazole. At 24 h, prolactin (PRL) secretion was 737±63 ng/ml in control cells. In cells treated with 0.1, 1 and 10µM: nocodazole for 24 h, PRL secretion was reduced to 200±30 ng/ml. After a 24 h incubation with the drugs, cells were washed with drug-free medium and challenged with 100NM: TRH for 10 min. TRH-stimulated PRL secretion was 35±7 ng/ml in control cells, 14±0.5 ng/ml in vinblastine-treated cells and 8.8±0.1 ng/ml in nocodazole-treated cells. [(3)H]TRH binding to GH(3) cell membrane was inhibited by about 15% by vinblastine and nocodazole. In vinblastine and nocodazole treated cells, polymerized tubulin levels decreased by 46 and 55%, respectively. These observations that vinblastine suppresses PRL secretion by GH(3) cells suggest that this drug might be useful as a therapeutic agent for prolactinomas.

The results of exposure to immobilization, hemorrhagic shock, and cardiac hypertrophy on beta-endorphin in rat cardiac tissue.

In the present study, beta-endorphin (BE), beta-lipotropin (B-LPH) and the ratio of beta-endorphin to beta-lipotropin (BE:B-LPH) were determined in rat cardiac tissue in response to physical stress induced by immobilization and cardiovascular stress resulting from hemorrhagic shock and pressure overload-induced cardiac hypertrophy. As compared with controls, BE was increased and B-LPH was decreased in cardiac tissue from animals subjected to immobilization, and there was also a significant rise in the ratio of BE:B-LPH. Cardiac BE remained unchanged following hemorrhage, while B-LPH was diminished, resulting in an increase in the ratio of BE:B-LPH. Similarly, the concentration of BE was unchanged, the concentration of B-LPH was significantly diminished and the ratio of BE:B-LPH was significantly increased in hypertrophied hearts. Thus, immobilization-induced stress, hemorrhagic shock, and cardiac hypertrophy all increased the ratio of BE:B-LPH in the heart. However, it appears that immobilization stress induces an increase in cardiac BE, whereas cardiovascular stress results in a preservation of BE in the heart and a reduction in cardiac B-LPH. The data suggests that physical stress (induced by immobilization) and cardiovascular stress (i.e., hemorrhage, hypertrophy) have differential effects on the synthesis of BE and the post-translational processing of proopiomelanocortin in the heart. Furthermore, the alterations in cardiac tissue BE and possibly B-LPH may play a role in the response of the heart to physical and cardiovascular stress.

Geriatric assessment teams in nursing homes: do they work?

Several studies have examined the effectiveness of geriatric assessment teams in outpatient and acute care settings. This project compared medical records of 69 consecutive nursing home patients randomly assigned on arrival to team (n = 33) and nonteam (standard care, n = 36) conditions. Quality-of-care indices and healthcare service utilization were compared over a 12-month postadmission period. Team patients had a significantly greater number of diagnoses and ancillary services combined with nonsignificant trends toward decreased mortality, fewer emergency department visits, and fewer drugs prescribed. The team approach improves quality of care. Additional clinical studies evaluating the effectiveness of geriatric assessment teams should be made in other nursing homes.

Characterization of beta-endorphin- and alpha-MSH-related peptides in rat heart.

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that beta-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to beta-endorphin- and alpha-MSH-related peptides. Ion exchange HPLC analysis revealed that beta-endorphin(1-31) was further processed to alpha-N-acetyl-beta-endorphin(1-31), which comprised 35.9 +/- 0.1% of total immunoreactivity, and smaller amounts of beta-endorphin(1-27), beta-endorphin(1-26), and their alpha-N-acetylated derivates. The predominant alpha-MSH immunoreactive peptides coeluted with alpha-MSH and N,O-diacetyl-alpha-MSH by reverse-phase HPLC, although small amounts of ACTH(1-13)-NH2 were also present. Thus, multiple forms of beta-endorphin and alpha-MSH are localized in rat heart. beta-Endorphin(1-31) is a minor constituent, however, indicating that nonopioid beta-endorphin peptides predominate.

Determination of total and free carbamazepine and the principal metabolites in serum by high-performance liquid chromatography with photodiode-array detection.

A precise and accurate high-performance liquid chromatography (HPLC) method has been established for the simultaneous analysis of carbamazepine (CBZ), carbamazepine-10,11-epoxide (CBZ-E), and trans-10,11-dihydroxy-10,11-dihydro-CBZ (CBZ-H) in serum samples and their ultrafiltrates. CBZ and its metabolites are eluted in a 3-microM ODS-Hypersil column (250 x 2 mm) at a column temperature of 40 degrees C. The mobile phase is a mixture containing potassium phosphate buffer-acetonitrile-methanol (110:50:30, vol/vol/vol) at a flow rate of 0.2 ml/min. Signals are monitored by a photodiodearray detector with a main sample wavelength of 215 nm and a bandwidth of 10 nm. Coefficients of variation (CVs) for within- and between-day are within 5%, with the recovery rates ranging from 98.16 to 104.64%. This method has the necessary sensitivity and linearity for routine therapeutic monitoring of both total and free CBZ and its principal metabolites. Total serum concentrations of CBZ, CBZ-E, and CBZ-H obtained from 55 epileptic children were 12.58 +/- 4.42, 2.45 +/- 1.22, and 5.83 +/- 3.17 (mean +/- SD, micrograms/ml), respectively. Levels of free CBZ, CBZ-E, and CBZ-H were 2.59 +/- 0.93, 1.05 +/- 0.57 and 3.73 +/- 1.87, respectively. Free fractions of CBZ, CBZ-E, and CBZ-H were 20.98 +/- 4.34, 42.63 +/- 8.21, and 65.41 +/- 7.80%, respectively. CBZ-H and CBZ-E had larger CVs than did CBZ (54.34 and 49.75 vs 35.15%, respectively, for total levels, and 50.31 and 54.46 vs 36.22%, respectively, for free levels), as well as higher free fractions. Determination of both total and free concentrations and free fractions of CBZ and its metabolites, as well as their ratios, should provide additional needed information for therapeutic drug monitoring of CBZ.

Simultaneous determination of carbamazepine, phenytoin, phenobarbital, primidone and their principal metabolites by high-performance liquid chromatography with photodiode-array detection.

We have established a precise and accurate high-performance liquid chromatographic method for the simultaneous assay of carbamazepine, phenytoin, phenobarbital, primidone and their principal metabolites. This method has been used for the analysis of these drugs and the metabolites in serum, saliva and urine samples. Acetonitrile is used for the deproteinization of serum and saliva samples while solid-phase extraction is utilized for urine sample pretreatment. Samples of 2 microliters are injected onto a 3-microns ODS-Hypersil column (250 mm x 2 mm I.D.) with a column temperature of 40 degrees C. The drugs and metabolites are eluted with a mobile phase containing potassium phosphate buffer-acetonitrile-methanol (110:50:30, v/v/v) at a flow-rate of 0.2 ml/min. Signals are monitored by a photodiode-array detector at a sample wavelength of 200 nm with a bandwidth of 10 nm. These four commonly used antiepileptic drugs and their six metabolites are well separated from one another within 15 min. Within-day coefficients of variation (C.V.) are within 5% in most cases and between-day C.V. are from 2.32 to 4.75%. The recovery rates range from 95.12 to 104.42%. This method has the necessary sensitivity and linearity for routine therapeutic monitoring of both total and free drug levels and may be employed for pharmacokinetics studies of drug interactions and metabolism as well.

Determination of free valproic acid: evaluation of the Centrifree system and comparison between high-performance liquid chromatography and enzyme immunoassay.

Valproic Acid (VPA) is an important drug for the treatment of several types of seizures because it has a wide spectrum of activity. Since VPA has an unusual nonlinear binding characteristic and a wide interindividual variation, monitoring of its free concentration can be helpful in patient management. The determination of unbound VPA is more difficult because an extra sample preparation step is needed and the concentration of free VPA is low. Free drug monitoring can assume a more important role if there is a refinement in the technology. A high-performance liquid chromatography (HPLC) method with isocratic elution has been established for the analysis of the 4-bromomethyl-7-methoxycoumarin (BrMMC) derivative of free VPA. This method has a better sensitivity, linearity, and precision than enzyme immunoassay (EIA). Ultrafiltration with the Centrifree system was evaluated for the sample preparation. The influence of centrifuge times, relative centrifugal forces, and the starting sample amounts on the final results of the ultrafiltration were investigated. There was a satisfactory correlation between the free VPA levels determined by the HPLC method and the concentrations obtained by EIA. The total and free VPA were determined on 100 samples from 36 patients. The total VPA levels were in a range of 25 to 208 micrograms/ml, free VPA concentrations ranged from 1.92 to 55.75 micrograms/ml with the free fractions from 7 to 37%.