Tumor necrosis factor-alpha as a biochemical marker of pain and outcome in temporomandibular joints with internal derangements.

OBJECTIVE: Previous studies have demonstrated the presence of tumor necrosis factor-alpha (TNF) in human temporomandibular joint (TMJ) synovial fluid. The present study continues the investigation of the role of TNF in TMJs with internal derangements. MATERIALS AND METHODS: Synovial fluid was obtained from 18 TMJs in 12 patients undergoing either arthroscopy (14 joints) or arthrotomy (four joints) for internal derangements. Standardized clinical data were collected preoperatively, intraoperatively, and postoperatively. RESULTS: When pain on palpation was absent, the mean preoperative TNF level was 14 +/- 6 ng/mL. When pain on palpation was present, the mean TNF level was 42 +/- 39 ng/mL (significant difference at P = .05). When the surgical outcome was poor, the mean preoperative TNF level was 26 +/- 9 ng/mL. When the outcome was within the stated guidelines for a favorable result, the mean TNF level was 12 +/- 7 ng/mL (significant difference at P = .05). In addition, a significant reduction (P = .05) in TNF following joint lavage (preoperative, 48 ng/mL to postoperative, 7 ng/mL) was found. CONCLUSIONS: The finding of a positive correlation between preoperative pain and TNF values suggests a biochemical basis for the origin of the pain associated with internal derangements. The relationship between preoperative TNF levels and surgical outcome suggests that the prognosis for surgery may be predicted by measuring biochemical markers of joint disease.

Immunomagnetic separation of tumor necrosis factor alpha. I. Batch procedure for human temporomandibular fluid.

A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.

Immunomagnetic separation of tumor necrosis factor alpha. II. In situ procedure for the human gingival space.

An in situ procedure has been developed for the separation of tumor necrosis factor (TNF) alpha directly from the fluid in the human gingival space. Paramagnetic beads coated with anti-TNF monoclonal antibodies were introduced into the gingival space of the subject with a polypropylene-tipped calibrated delivery system and retrieved using a permanent magnet designed to fit into the space. After retrieval, the amount of immunoadsorbed TNF was quantified using an immunochemical assay called the "cluster assay". The results indicate that following the appropriate preparation of the site, over 95% of the beads could be recovered. With this method we found that 62% of those cavities sampled contained TNF and that the values ranged from 0.10 to 13.0 ng/ml with a mean value of 1.7 ng/ml. A comparison of these values with those obtained from the same space using other methods suggests that the immunomagnetic method was more effective in retrieval of TNF. Because the separation is performed in situ we have named the procedure "chromatobiosis".

Determination of cefsulodin, cefmenoxime, and cefadroxil as residues on surfaces.

Cefsulodin, cefmenoxime, and cefadroxil are degraded instantaneously in aqueous sodium hypochlorite, sodium hypochlorite-detergent, or alkaline detergent solutions. These alkaline solutions are used to clean surfaces that have been exposed to the cephalosporins. The cleaned surfaces are monitored for residual drug levels (microgram) using a wet swab-dry swab technique. After extraction from the swabs, the content of the respective cephalosporin is determined in the solution by high-performance liquid chromatography. The limit of detection for each of the compounds is 0.1 microgram/ml. Recoveries from nonporous surfaces ranged from 56 to 102%.

An unusual phosphatidylinositol turnover pathway in noradrenaline-perfused rat hearts.

1. The phosphatidylinositol turnover pathway has been studied in noradrenaline-perfused rat hearts using anion-exchange high performance liquid chromatography. 2. The active calcium-releasing compound inositol-(1,4,5)trisphosphate was detected together with its degradation products inositol-(1,4)bisphosphate and inositol monophosphate. All these products increased in response to noradrenaline stimulation. 3. At noradrenaline perfusion times from 5 s to 20 min there was no appearance of inositol-(1,3,4,5)tetrakisphosphate or its degradation products: inositol-(1,3,4)trisphosphate or inositol-(1,3) and (3,4)bisphosphates. 4. These data suggest the absence of the inositol-(1,4,5)trisphosphate phosphorylation/dephosphorylation pathway in heart.

Angiotensin II-stimulated phosphatidylinositol turnover in rat adrenal glomerulosa cells has a complex dependence on calcium.

The stimulation of phosphatidylinositol (PI) turnover by angiotensin II in rat adrenal glomerulosa cells has been studied in detail and shown to have a complex dependence on Ca2+. After the addition of angiotensin II, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate increased rapidly and transiently. The transient increase was followed by a slower sustained rise, which continued for up to 30 min. Inositol phosphate accumulation during the sustained phase was decreased when experiments were performed in Ca2+-free medium. The initial transient response was not altered. Addition of the Ca2+ ionophore A23187 enhanced the angiotensin II response at 20 min, but not the 15 sec response. The sustained response, but not the transient response, was attenuated by the Ca2+ channel blocker nifedipine, indicating that the effect of Ca2+ required uptake through voltage-dependent Ca2+ channels. Subsequent studies showed that cAMP decreased inositol phosphate accumulation at 20 min while having no effect at 15 sec. Also, incubation with phorbol 12-myristate 13-acetate produced a more effective inhibition of the sustained response than of the initial transient response. However, while the transient and sustained phases of PI turnover were differently affected by Ca2+ and inhibitory compounds, the profiles of inositol phosphates generated were similar. At both 15 sec and 20 min inositol-(1,4,5) trisphosphate was detected, indicating sustained cleavage of PI-(4,5) bisphosphate. Taken together, the results suggest that while the initial PI turnover response is independent of Ca2+ and presumably initiates the rise in cytosolic Ca2+, sustained response requires entry of Ca2+ to maintain elevated cytosolic Ca2+ concentrations. Thus, while the increase in cytosolic Ca2+ may have a direct role in the stimulation of aldosterone synthesis, it is also required to sustain the PI turnover response to angiotensin II.

Stimulation of phosphatidylinositol metabolism in the isolated, perfused rat heart.

Receptor-stimulated phosphatidylinositol turnover has been studied in isolated, perfused, [3H]inositol-labelled rat hearts by measuring accumulation of inositol phosphates in the presence of lithium chloride. Inositol phosphate accumulation was stimulated by norepinephrine (3 X 10(-5) M) and carbachol (10(-3) M), the increases averaging from 931 +/- 59 (n = 6, mean +/- SEM, cpm/g heart) to 4,165 +/- 609 (n = 6, p less than 0.01) for norepinephrine and to 1,853 +/- 354 (n = 6, p less than 0.05) for carbachol. The norepinephrine stimulation was antagonized by prazosin (10(-7) M) but not by propranolol (10(-7) M), indicating mediation via alpha 1-adrenoceptors. The carbachol stimulation was antagonized by atropine (10(-7) M). The stimulation by norepinephrine was significantly higher in right atria (837 +/- 151 to 6,614 +/- 1,210, n = 6, cpm/g tissue) than in other regions of the heart. Both norepinephrine and carbachol stimulated the formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate with norepinephrine stimulation being detected as early as 15 seconds. Furthermore, the inositol trisphosphate was identified as the -1,4,5 isomer by anion exchange high-performance liquid chromatography. These data are consistent with the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate yielding inositol-(1,4,5)-trisphosphate. Inositol-(1,3,4)-trisphosphate was not detected in heart preparations, suggesting unusual metabolism of inositol-(1,4,5)-trisphosphate in heart tissue.

Isolation, amino acid sequence and action of guinea-pig ACTH on aldosterone production by glomerulosa cells.

Though minor sequence differences between-species have been reported for adrenocorticotrophin, ACTH(1-39), the steroidogenic moiety ACTH(1-24) has appeared invariant in mammals. We here report the isolation, purification and amino acid sequencing of guinea-pig (GP) ACTH in which Pro24 is replaced by Ala24, and the demonstration that GP-ACTH stimulates aldosterone production to maximal levels well above those seen with human ACTH(1-39) or Synacthen, ACTH(1-24) amide, the synthetic ACTH fragment widely used for diagnostic and therapeutic purposes.

Evidence for a lack of inositol--(1,4,5)trisphosphate kinase activity in norepinephrine-perfused rat hearts.

The products of the phosphatidylinositol turnover pathway in norepinephrine-perfused hearts have been examined using high performance liquid chromatography. Inositol-1 phosphate, inositol-(1,4)bisphosphate and inositol-(1,4,5)-trisphosphate were all increased in response to norepinephrine stimulation. However, at perfusion times from 5 sec to 20 min there was no appearance of inositol-(1,3,4,5)tetrakisphosphate or inositol-(1,3,4)trisphosphate. This suggests the absence of the inositol-(1,4,5)trisphosphate phosphorylation/dephosphorylation pathway in heart and demonstrates that this secondary pathway is not essential to the functioning of the phosphatidylinositol turnover cycle.

Determination of acetazolamide in biological fluids by reverse-phase high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of acetazolamide in whole blood, plasma, and urine was developed. Samples of biological fluids containing various concentrations of acetazolamide were spiked with the internal standard, sulfadiazine. Samples were then mixed with a 50% ammonium sulfamate solution. Whole blood samples were heated for 25 s in boiling water. All samples were extracted with ethyl acetate; a phosphate buffer (pH 8.0) was used to wash the extracts. Acetazolamide was back-extracted into a glycine buffer (pH 10.0), which was then washed with ether. Separation of acetazolamide and internal standard from other biological constituents was achieved on a 10-micron C18 reverse-phase column using an acetonitrile-methanol-acetate buffer (pH 4.0). The eluant was monitored at 254 nm. All calibration curves were linear, and the results from reproducibility studies were excellent. Application of the method to human pharmacokinetic studies was demonstrated.

Observations on lithium disposition in the elderly.

The disposition kinetics of lithium were studied in six elderly women who had been maintained on this medication for several years. Lithium administration was stopped abruptly. Plasma, whole blood, and erythrocyte half-lives were then measured, as well as renal plasma clearance and distribution volume. Half-life values in whole blood and in its components were found to be similar. The plasma lithium half-life approximated that in younger schizophrenics, but appeared prolonged when compared with half-lives reported for young normal volunteers. Renal plasma clearance and distribution volume of lithium appeared substantially reduced when compared with published values. The data are consistent with altered lithium disposition in the aged.

Utilization of intravenously administered glucose-oligosaccharides in growing miniature pigs.

The ability of intravenously administered glucose or glucose oligosaccharides to supply 12% of the energy requirement of the growing miniature pig was determined. All pigs were fed a stock diet from days 5-28 of life, and a central venous catheter was inserted at 29 days. For the next 30 days, all pigs were fed both enterally and parenterally. Positive control pigs were fed the stock diet with infusions of an isotonic balanced electrolyte solution. Negative control and test animals were fed a diet containing cellulose to replace 12% of energy. Negative control animals received the balanced electrolyte solution intravenously, while test animals received either intravenous glucose of glucose oligosaccharides at levels providing 12% of energy. Carbohydrate utilization was measured by urinary loss of carbohydrate, as well as by growth and body composition. Chemical analyses of the urine indicated utilization of both glucose and glucose oligosaccharides. Glucose oligosaccharides were utilized less well (85%) than glucose (greater than or equal to 99%). No statistically significant differences in weight gain were noted between groups, although weight gain in negative control animals and those animals infused with glucose oligosaccharide solutions were lower than those in the other groups. Body fat analyses also indicated that glucose oligosaccharide solutions were less effective than glucose as an energy source.

Simultaneous determination of hydrochlorothiazide and triamterene in capsule formulations by high-performance liquid chromatography.

A stability-indicating, high-performance liquid chromatographic method is presented for the simultaneous determination of hydrochlorothiazide and triamterene in two-component capsule formulations. An aliquot of the sample is dissolved in a mixture of acetonitrile and aqueous acetic acid, mixed with m-hydroxacetophenone as an internal standard, and chromatographed on octadecylsilane bonded to microparticulate silica using 80% acetate buffer (pH 5.0, 0.004 M)-15% acetonitrile-5% methanol as the mobile phase. The relative standard deviations are +/- 1.1-1.6% for hydrochlorothiazide and +/- 1.7-2.2% for triamterene for two formulations. Recoveries of the two drugs added to placebos ranged from 98.4 to 101.7%.

Hepatotoxicity following enflurane anesthesia.

We present a patient who developed clinical, biochemical, and histologic evidence of severe hepatic injury following enflurane anesthesia.