Pathogenesis of calcium crystal deposition in the ligamentum flavum correlates with lumbar spinal canal stenosis.

OBJECTIVE: To investigate the histological and immunohistochemical properties of degenerative changes and calcium crystal deposition in the lumbar ligamentum favum. METHODS: We examined the ligamentum flavum harvested from 119 surgical cases with symptomatic lumbar spinal stenosis. Sections of the ligament were examined by scanning electron microscopy (SEM), energy dispersive X-ray micro-analysis, and were immunostained for S-100 protein, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and CD34. The results were compared with those of ligament tissue harvested from 10 cases of lumbar disc herniation. RESULT: The elastic fibres of the ligamentum favum showed regular, or sometimes irregular, and fragmented fibre bundles. Large areas of fibrosis with reduced elastic component and increased collagenous tissue were frequently seen in the degenerated ligaments. Calcium crystal deposits were observed in these fibrous ligaments, associated with many hypertrophic chondrocytes, and with small blood vessel formation. These chondrocytes stained positively for S-100 protein, VEGF and bFGF Calcium pyrophosphate dihydrate crystals were identified in the calcium deposit area. CONCLUSION: We believe that rupture of elastic fibre bundles is the first change to occur in degeneration of the ligamentum favum. Calcium crystal deposition was seen within these fibrous and chondrometaplastic areas. Hypertrophic chondrocytes regulate crystal formation and tissue reconstruction by secreting cytokines.

A toxic reagent-free method for normal-phase matrix solid-phase dispersion extraction and reversed-phase liquid chromatographic determination of aldrin, dieldrin, and DDTs in animal fats.

A method for the determination of aldrin, dieldrin, DDT, DDE, and DDD contamination in animal fats (beef tallow, lard, and chicken fat) without using toxic reagents is developed, that uses high-performance liquid chromatography after the sample has been prepared by matrix solid-phase dispersion (MSPD) with acidic alumina oxide. A reversed-phase C(1)-silica column with a mobile phase of 50% (v/v) ethanol solution (in water) and a photo-diode array detector were used for the determination. Average recoveries of the target compounds (0.2-5.0 microg g(-1)) ranged from 84-98%, with coefficients of variation of <5%. The limits of quantitation were

Transferring and distributing profiles of p,p'-(DDT) in egg-forming tissues and eggs of laying hens following a single oral administration.

Laying hens were administered orally with a single dose of p,p'-(DDT) (1 mg/kg bodyweight). The concentrations (microg/g) of DDT or its metabolites, p,p'-(DDE) and p,p'-(DDD), in the main tissues involved in egg formation (blood, liver, ovary, and oviducts) and egg yolk, collected 1 day after DDT dosing, were determined by normal-phase high-performance liquid chromatography. The limits of detection were 0.04 microg/g for DDT, 0.07 microg/g for DDE and 0.06 microg/g for DDD. In extractable fats from the above tissues and egg yolk, DDT and DDE were transferred/distributed throughout the tissues and egg yolk. DDD was detected only in the liver. The findings indicate that DDT is metabolized instantaneously to DDE/ DDD in the hen's body and they are transferred rapidly into the egg-forming tissues and egg yolk. Among the four tissues and yolk fats examined, the DDT levels were high in the ovary, oviduct and egg yolk; the DDE levels were high in the liver, ovary and oviduct and lowest in the yolk (P < 0.01).

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues.

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP-4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.

Genotoxicity of gardenia yellow and its components.

Gardenia fruit (Gardenia jasminoides ELLIS) is widely used as a natural food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. "Gardenia yellow" is a natural food colorant which is extracted by ethanol from gardenia fruit. The purpose of this study was to evaluate the genotoxicity of gardenia yellow. Genotoxicity of gardenia yellow and its components, crocetin, gentiobiose (a component of crocin), geniposide and genipin (formed by hydrolysis of geniposide), was studied by Ames test, rec-assay, and sister chromatid exchange (SCE) using V79 cells. Gardenia yellow and its components were found not to be mutagenic in the Salmonella reverse mutation assay. Gardenia yellow and genipin caused damage of DNA in rec-assay. Gardenia yellow induced a significant dose-dependent increase of SCE frequency (8.6 times at 1000 microg/ml as the value for the solvent control). Only genipin induced SCEs significantly among the components of gardenia yellow. Moreover, genipin induced a significant increase of tetraploids at all doses tested (95% at 8 microg/ml). Gardenia yellow preparation was analyzed by capillary electrophoresis (CE), and geniposide was detected. However, genipin was not observed. In conclusion, we have shown that genipin possesses genotoxicity. Furthermore, there were unidentified genotoxicants in gardenia yellow.

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues.

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.

Transfer and distribution profiles of dietary sulphonamides in the tissues of the laying hen.

The five sulphonamides, sulphadiazine (SDZ), sulphadimidine (SDD), sulphamethoxazole (SMX), sulphamonomethoxine (SMM) and sulphaquinoxaline (SQ), were fed to laying hens at a dietary concentration of 100 mg kg(-1), respectively. On the 7th day after the start of feeding, the drug concentrations in the plasma, muscle and the main tissues involved in egg formation, the liver, and ovary and oviducts (magnum and isthmus plus shell grand) were determined by high-performance liquid chromatography. Dietary sulphonamides were distributed throughout the above tissues. SQ was found at the highest concentration in all tissues, while the reverse was true for SDD. The ratio of SDD concentrations in the main tissues involved in egg formation to that in the plasma were greater than those for the other drugs.

In vitro hepatic biotransformation of aldrin and dieldrin in food-producing animals.

The hepatic biotransformation of aldrin (AD) and dieldrin (DD) was studied in liver post-mitochondrial supernatants (S-9s) from laying hens, female cattle and swine. S-9s were incubated with 0.03 nmol of AD or DD for 1 h. After 1 h, AD in the samples was almost epoxidated to DD. This formation was found with all the animal S-9s, and the highest rates occurred in pig S-9 (P < 0.01), followed by cow and hen S-9s. No reduction of DD was found with any of the S-9s.

Determining the procedure for routine residue monitoring of sulfamethazine in edible animal tissues.

A simplified method to determine/identify residual sulfamethazine (SMZ) in edible tissues from cattle, pigs, chickens and sheep by a high-performance liquid chromatography (HPLC) with a photo-diode array detector is presented. The sample preparation was performed by homogenizing with 30% (v/v) ethanol in water followed by an Ultrafree-MC/PL as a centrifugal ultra-filtration unit. For determination/identification of SMZ, a reversed-phase C(4) column and a mobile phase of 15% (v/v) ethanol in water with a photo-diode array detector was used. Average recoveries from spiked SMZ (0.1-1.0 mg kg(-1)) were >or=80% with coefficients of variation between 1.3 and 4.3%. The limits of quantitation were calculated to be 0.057-0.060 mg kg(-1). The total time and solvent required for the analysis of one sample were <40 min and <2 mL of ethanol, respectively.

Herniation of cervical intervertebral disc: immunohistochemical examination and measurement of nitric oxide production.

STUDY DESIGN: Surgically obtained cervical herniated intervertebral discs were examined histologically and immunohistochemically. The production of nitric oxide (NO) in the local tissue was examined using the electron spin resonance (ESR) method. OBJECTIVES: To investigate the local histologic and immunohistochemical changes in cervical disc herniation, including NO production, and to compare such changes with those in autopsy cases. SUMMARY OF BACKGROUND DATA: Very little is known about the histopathologic processes of cervical disc herniation. In addition, no information is available on the level of in vivo NO production in cervical disc herniation. METHODS: Thirty-six herniated cervical discs obtained from 31 patients were immunohistochemically examined for localization of blood vessels, matrix metalloproteinase (MMP)-3, and inducible NO synthetase (iNOS). We also compared the production of NO, measured by the ESR method, in eight specimens with that of five control discs obtained from fresh cadavers. RESULTS: The presence of herniated discs correlated with the degeneration of cartilaginous endplate and torn anulus fibrosus. Formation of new blood vessels around the herniated discs was detected, using von Willebrand factor antibody, in seven uncontained hernias and 20 contained hernias. Immunohistochemical studies showed the presence of cells positive for MMP-3 (chondrocytes), iNOS (chondrocytes and granulation tissue) in cervical disc hernias. ESR analysis showed a significantly higher NO production in herniated cervical discs than in disc samples of fresh cadavers. CONCLUSIONS: Herniated cervical intervertebral disc is characterized by the presence of an inflammatory process associated with neovascularization and increased expression of MMP-3. Production of NO was markedly high in both contained- and uncontained-type hernias.

Normal-phase high-performance liquid chromatographic determination and identification of aldrin, dieldrin, and DDTs in eggs.

A method for the routine monitoring of residual aldrin, dieldrin, pp'-DDT, op'-DDT, pp'-DDE, and pp'-DDD in eggs by high-performance liquid chromatography (HPLC) with a photo-diode array (PDA) detector is described. The lipids extracted from a whole egg are cleaned by a solid-phase extraction (SPE) ISOLUTE NH2 column with a diethyl ether-hexane (5:95, v/v) eluent. The HPLC separation is carried out using a normal-phase (LiChrosorb NH2) column, a heptane-hexane eluent (97:3, v/v), and a PDA detector. The average recoveries from fortified target compounds (0.1, 0.2, 0.3, and 0.4 microg/g, respectively) are > or = 83%, with standard deviations (SDs) between 2 and 5%. The interassay variabilities and their SDs are < or = 4.8% and < or =0.7%, with intra-assay variabilities of 2.1-3.3%. The limits of determination for these compounds range from 0.04 to 0.08 microg/g.

In vitro hepatobiotransformation of sulphadimethoxine in laying hens.

The biotransformation of sulphadimethoxine (SDM) was estimated in liver post-mitochondrial supernatants (S-9) from laying hens. The pathway and activity for hen S-9 were compared with those for cow and pig S-9. The formation of the hydroxylation of SDM, 6-hydroxy SDM (6-OH-SDM), was found only with hen S-9. The N4-acetylating activity of SDM in hen S-9 was lower than in cow and pig S-9. All S-9 from the three species de-acetylated N4-acetyl SDM (N4-AcSDM). With respect to the hydroxylation and N4-acetylation rates in hen S-9, the values incubated at 41 degrees C were higher than those at 37 degrees C (P < 0.01).

Decreasing half-life of dieldrin in egg yolk following a single oral administration of aldrin to laying hens.

Laying hens were treated orally with a single dose of aldrin (AD) 1 mg/kg body weight. Concentrations (microgram/g) of AD or its epoxide (= dieldrin, DD) in the yolk of eggs laid for 21 days after AD treatment were determined by normal-phase high-performance liquid chromatography. The limits of determination were 0.02 microgram/g for AD and 0.03 microgram/g for DD, respectively. After AD treatment, although the low levels of AD (mean 0.02-0.03 microgram/g) were observed only during a three-day period (from 4th to 6th days), DD (mean 0.15 microgram/g) was found already on the 2nd day, indicating that the epoxidation of AD to DD in the hen's body is rapid. The highest level of DD (mean 0.40 microgram/g) was detected on the 6th day, and then DD levels decreased slowly and were detected up to the 21st day. In this decreasing phase, the half-life of DD in the yolk was estimated to be 25.6 days with a 95% confidence interval from 22.7 to 29.4 days.

High-performance liquid chromatographic procedure for routine residue monitoring of seven sulfonamides in milk.

A simplified method for routine monitoring of 7 residual sulfonamides (SAs) (sulfadiazine (SDZ), sulfamerazine (SMR), sulfadimidine (SDD), sulfamonomethoxine (SMM), sulfamethoxazole (SMX), sulfadimethoxine (SDM), and sulfaquinoxaline (SQ)) in milk using high-performance liquid chromatography (HPLC) with a photodiode array detector is described. The spiked and blank samples were cleaned up by using an Ultrafree-MC/PL centrifugal ultrafiltration unit. For determination/identification, a Mightysil RP-4 GP column and a mobile phase of 25% (v/v) ethanol in water with a photodiode array detector were used. Average recoveries from milk samples spiked with 0.05, 0.1, 0.2, and 0.5 microg mL(-1) of each drug were >82%. The inter- and intra-assay variabilities were 2.0-3.1%. The practical detection limits for 7 SAs were 0.005-0.02 microg mL(-1). The total time and amount of solvent required for the analysis of one sample were <40 min and <6 mL of ethanol, respectively. No toxic solvents were used.

Matrix solid-phase dispersion extraction and high-performance liquid chromatographic determination of residual sulfonamides in chicken.

Simultaneous determination of the six sulfonamides (SAs) sulfadiazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline in chicken using matrix solid-phase dispersion (MSPD) with neutral aluminium oxide as an MSPD sorbent and high-performance liquid chromatography (HPLC) is presented. In the present MSPD, six SAs could be isolated by only one step, elution with a 70% (v/v) aqueous ethanol solution, without the sorbent conditioning and the sorbent-tissue matrix washing. For the HPLC determination, a LiChrospher 100 RP-8 and a mixture of 1% acetic acid solution (pH 3.0, in water)-acetonitrile-N,N-dimethylformamide (78:22:5, v/v/v) as the mobile phase with a photodiode array detector were used. Average recoveries were greater than 87.6% with relative standard deviations between 0.5 and 8.6%. The total time and amount of solvent required for the analysis of one sample were <1.5 h and <12 ml, respectively.

Transference of dietary veterinary drugs into eggs.

Twelve veterinary drugs, bacitracin (BC), chloramphenicol (CAP), chlortetracycline (CTC), oxytetracycline (OTC), tylosin (TS), amprolium (APL), furazolidone (FZD), nicarbazine (NCZ), ormetoprium (OMP), sulfadimidine (SDD), sulfadimethoxine (SDM) and sulfamonomethoxine (SMM) were fed to laying hens for 14 days, each at a dietary concentration of 500 mg/kg. The concentrations of the drugs in the eggs from these birds were determined at 2-day intervals for 14 days after the start of feeding. The relationship between the concentrations of the drugs (mg/kg) in the eggs and the number of days after the start of feeding was analysed by regression and covariance analyses. The concentrations of the drugs in the eggs became constant after 4 days for OTC, TS, FZD and all the sulfonamides, and after 6 days for CAP, APL, NCZ and OMP. No BC or CTC was detected in the eggs. The transfer rates of the 10 drugs (excluding BC and CTC) from the feed to eggs varied from 0.005% for TS up to 1.540% for SDD.

Polluting profiles of dieldrin and DDTs in laying hens of Osaka, Japan.

Contaminating/accumulating levels of organochlorine pesticides in extractable fats from a basal diet, eggs and seven tissues (adipose tissue, blood, kidney, liver, muscle, ovary and oviduct) and excreta of laying hens that were kept in a general poultry farm of Japan were examined. No benzene hexachlorides or aldrin was detected [< 1 part per billion (p.p.b.)] overall. Dieldrin and all DDTs (p,p'-DDE, o,p'-DDT, p,p'-DDD and p,p'-DDT) were contaminated in the dietary fats (mean range 10-70 p.p.b.). Although dieldrin (4-58 p.p.b.), p,p'-DDE (65-196 p.p.b.) and p,p'-DDT (30-73 p.p.b.) were found to be accumulated in all the tissue fats and egg yolk fats, they were not detected in the dried excreta. Accumulations of o,p'-DDT and p,p'-DDD were found only in the liver fat (92 p.p.b.) and in the kidney fat (27 p.p.b.), respectively. In all the samples, p,p'-DDE levels were highest in comparison with the other compounds. For all organochlorine pesticides detected, the accumulated levels were well below the practical residue limits.

Spontaneous remission of a solitary intraspinal synovial cyst of the lumbar spine.

We report on a 15-year-old boy in whom a spontaneous remission of a symptomatic synovial cyst, possibly emanating from the L4-5 facet joint, was noted. The medical history suggested that sport-related overactivity and/or minor trauma was the underlying cause. Conservative treatment for several months may be one treatment option if the cyst wall is not calcified and the symptoms and signs related to radiculopathy show a gradual decrease.

Simplified determining procedure for routine residue monitoring of sulphamethazine and sulphadimethoxine in milk.

A simplified determining/identifying method for residual sulphamethazine (SMZ) and sulphadimethoxine (SDM) in milk by using a high-performance liquid chromatography (HPLC) with a photo-diode array detector was presented. Both sulphonamides in cow's milk samples were extracted by only stirring with ethanol followed by an Ultrafree-MC/Biomax as a centrifugal ultra-filtration unit. For determination/identification of SMZ and SDM, a Mightysil RP-18 GP Aqua column and a mobile phase of 25% (v/v) ethanol solution (in water) with a photo-diode array detector was used. Average recoveries from spiked SMZ and SDM (10-1000 ng/ml each drug) were > or = 83% with the relative standard deviations between 1.4 and 3.7%. The limit of quantitation (LOQ) were calculated to be 5 ng/ml for SMZ and 10 ng/ml for SDM, respectively. The values were below the MRL/tolerance (SMZ, 25 ng/ml; SDM, 10 ng/ml). The total time and solvent required for the analysis of one sample were <35 min and <2 ml of only ethanol, respectively. No toxic solvents were used. The developed procedure was harmless to the human and environment.

Binding profile of spiramycin to oviducal proteins of laying hens.

In vitro protein binding of spiramycin (SP) in the plasma and oviducts of laying hens was studied. The data for SP were compared with those for oxytetracycline (OTC), sulphadimidine (SDD), sulphamonomethoxine (SMM) and sulphaquinoxaline (SQ). The two oviduct segments, magnum (M) and isthmus plus shell gland (IS), were collected. The soluble (cell sap) fractions from the magnum (M-S9) and the isthmus plus shell gland (IS-S9) were used as samples. Plasma protein binding was highest for SQ (81.4%) (P < 0.01), and lowest for SDD (30.9%) (P < 0.01). No M-S9 protein binding of OTC was found. The IS-S9 protein binding of SP (60.4%) was very much higher than those of OTC (0.8%), SDD (4.1%), SMM (4.0%) and SQ (12.3%) (P < 0.01). Biological half-lives of these drugs in egg albumen were directly correlated to the extent of their binding to IS proteins. Of plasma, M-S9 and IS-S9, variation in SP concentration in the ranges from 1 to 20 micrograms/ml did not alter the binding properties of the drug.