| Background:Tuberculosis was an old contagious disease and it was the longest disease accompany with human being, there was tuberculosis in the new stone age(BC.500-10000 years) people cervical vertebra fossil which unerthed on heidelberg in 1904. eighteen century medius, the tuberculosis mortality rate was 900/10 ten thousand, hitherto the number of world people to lost their life becouse of tuberculosis reach to 2 hundred million.. tuberculosis was called "white pest", adding to warfare and penury, deteriorated the condition and accelerated the dissemination of tuberculosis even to death, deduced a series of tragedy in human history. up to now tuberculosis was still the most severe contagious disease in developing country. In our country this so called "Phthisis" diseas once deprived numerous life of labourer in history. Before republic of china established there were "nine can not survived in ten phthisis" in civilian we could see the rampant condition at that time and the terror becouse of tuberculosis. When streptomycin and other antituberculosis drugs one after another emerged, adding to government thought highly of prevention and cure works of tuberculosis after liberation, tuberculosarium, prophylactico-therapetic institution and research instituteb incessant setted up all over the country. Our orthopedics ancestor MR. Fang Xian-Zhi firstly developed focus clearance of tuberculosis of bones and joints, made cure rate of tuberculosis of bones and joints incline remarkably. After twenty century 1970 years later, as our country developing and reforming we had make a large success in economic construction. The level of civilian living had been boosted tremendously, these made uberculosis fatality rate and infection rate incline rapidly. The cases of tuberculosis lead to some prevention and cure branch of tuberculosis close their door or modify to other medical therapy unit. But in recent ten years as infection of HIV and other disease of immune system developing, tuberculosis trended to rekindling globally. Now all the world there are 2000 ten thousand patients of tuberculosis, increase 800 ten thousand every years, In 1993 WHO proclaimed that we come into global emergency condition of tuberculosis. Now in our country the number of patients with tuberculosis is the secend in the world, the first in all infection disease. Bones and joints tuberculosis is the primary one of the out-pneumonophthisis amone the all tuberculosis tuberculosis, currently the are 30 ten toousand patients with bones and joints tuberculosis, once disappeared "Phthisis" emerge again. All of these alarm medical workers and ask a new requisition.Surgery as one stage in the whole procedure of treating tuberculosis, on the base of chemotherapy make surgery to clear focus of tuberculosis, we can remove pathologic vertebra driectly and make good use of drugs penestrating foucus, shorten course of treatment, enhance the cure rate of tuberculosis. On one hand surgery could clear the focus thoroughly, on the other hand it could incease blooding and trauma, then the postoperative complications happened more. It is a concerned problem to most surgeon that whether we can grasp the range which wait for surgery to remove, but few pharmacokinetic studies and clinical reports so far about the concentrations of antituberculosis drugs and their metabolite in vertebral foci have been reported. This make a difficult problem to define the range.Our manuscript use high-pressureliquid chromatography (HPLC) method to determine the concentrations of isoniazid, rifampicin, pyrazinamide and their metabolite in pathological vertebrae,serum as well as the normal osseous tissue, discuss the disposition of antituberculosis drugs in skeletal tissue of spinal tuberculosis after regular chemotherapy, in order to provide the theoretical basis for the improvement in treating spinal tuberculosis and modification in chemotherapy program.objectives.Study the the concentrations of antituberculosis drugs and their metabolite in skeletal tissue of spinal tuberculosis, to provide the program of chemotherapy and surgical treatment of spinal tuberculosis.Methods.1. investment object:Totally thirty-six patients with spinal tuberculosis, treated by consecutive ultrashort-course chemotherapy in conjunction with partial excision of pathologic vertebrae we investigated. There were 15 males and 21 females whose ages ranged from 23 to 75 years, with mean of 47.1 years. cervical vertebrae 2 cases, thoracic vertebrae 5 cases, thoracic waist vertebrae 10 cases, lumbar vertebrae 16 cases, Lumbosacral vertebrae 3 cases (table 3). According to Jain et al.7 CT classification and whether or not the sclerotic wall was formed around the foci among 36 patients, 13 cases were sorted into sclerotic group with the radiographic characters of vertebral sclerosis around the foci (Figure 1),23 cases divided into non-sclerotic group (Figure 2). The chemotherapeutic program that all patients abided was 2HRZE/8H2R2E2(toltal 10 months), which was recommended by the guidelines for national programmes of treatment of tuberculosis formulated by WHO. All of the patients have definite surgical indications, including spinal cord compression or neurological deficit, vertebral body collapse or kyphosis, wide paravertebral abscess or sinuses unresponsive to medical treatment and so on. All cases weigh mean of 57.5kgs, had normal preoperative hepatic and renal function, had no anti tuberculosis chemotherapy 3 months before hospitalization.25 of the patients who had greater bone destruction were treated with anterior or anteriorlateral (no open membrana pleuralis) approach debridement, bone grafting, fusion and anterior instrumentation,6 patients who had less bone destruction or poor medical condition were treated with posterior approach debridement and transpedicular instrumentation that was supplemented with posterolateral bone fusion and chemotherapy. All patients accoding to guidelines for national programmes:2HRZE/8H2R2E2, everday morning oral taken INH 0.3,RFP 0.45,SM 0.75,PZA1.5 to supervise and direct. After four weeks later when the toxic symptom alleviated, ESR<60 mm/h, HGB>100g/L the surgery would be taken.2. laboratory detection:①sample collectionAll cases underwent 4 weeks chemotherapy before surgery, on the day of surgery the antituberculosis drugs were administered as usual early in the morning. With the cooperation of the operators and the anesthetists, the process of drugs intake and the time recording as well as the samples' obtaining were under strict control. All samples were obtained at 100-120 min after drug intake, otherwise the concentration of drugs can hardly be got because the half-life of INH is only 120-180 min. when concentration level of antituberculosis drugs in blood exceed to their limit of quantitation (LOQ) and limit of detection (LOD) we cannot detect them. The vertebral samples were obtained from pathologic vertebrae, including sclerotic wall outside the foci, sequester and cheesy necrosis tissue inside the foci in sclerotic group and the foci in non-sclerotic group. Subnormal vertebra tissue (according to far or near foci 4mm sorted to A and B segment) (Figure 3), simultaneouly the blood samples were obtained by cruise nurses and self-contrast bone was obtained from ilium bone graft in both groups.②sample preperation disposalThe blood was centrifuged for 15 min at 3000 g. and the serum was collected for further determination. Pathologic vertebral tissues and ilium were immediately rinsed with normal saline for removing their adherent blood, then blotted dry, and placed in liquid nitrogen for the advanced procedure; To prepare the soft tissue and bone samples for drgus extraction, samples were cryodesiccated and pulverized into a fine powder using a SPEX 6870 freezer mill,then weighed 200μg with an analytic electronic balance and added 2ml methanol, centrifuged again, extracted supernatant for high-performance liquid chromatography (HPLC). All serum, soft tissue and bone samples were subsequently stored below-200℃until analysis.③Chromatographic condition:chromatographic column was shim-pack HRC-C8 (250mm×4.6mm,5μm) and guard column was Eclipse XDB-C18(125mm×4.6mm), The best separation was produced by using the mobile phase consisting of 0.02mol/L sodium heptanesulfonate(pH of 3.10)-CAN-MeOH (78:5:17) 6min,(20:5:75)16min, (78:5:17)27min with a flow rate of 0.8 ml/min and a detection wavelength of 254nm. All reagents employed were of HPLC grade and were purchased from Sigma (St. Louis, MO). Acetylisoniazid (Ac-INH) and 25-Desacetylrifampicin (De-RFP) standard preparation were prepared according to literature. Calibration curves were constructed according to peak area as ordinate and concentration as abscissa (Figure 4).1000μl methanol were taken in 200μl serum to precipitated protein and extracted supernatant after centrifuge, blowed with nitrogen gas to a final volume of 200μl. Vertebrae tissues were extracted accomplished using the extract with dimethylcarbinol- dichloromethane (1:1), extracted 200μl vertebrae tissues extract then disposed the same as serum. Sample injection volume was 20μl. From the resulting supernatant(Figure 5), a 200-μl aliquot was used for hemoglobin (Hgb) determination; the hemoglobin in the blood samples was determined using the cyanide method and in the bone extraction with the benzidine-superoxide method. For correction of blood contamination in the bone, the relevant method as described by Roncoroni et al.Results.1.15 cases of sclerotic group sample:2.13 cases of non-sclerotic group sample: Statistical analysisThe differences of concentration level of INH, RFP, PZA in singulorum fraction were analyzed respectively in two groups. Analyses were executed by SPSS statistical software, version 13.0 (SPSS UK Ltd). Data are expressed as mean±SD (standard deviation). Differences of means within groups were evaluated statistically by one-way ANOVA and Dennett t test. P value<0.05 was considered significant.BloodThe concentrations of three drugs and their metabolite in blood were as chartl,chart2.The concentration level of all drugs and their metabolite were higher than that of ilium, subnormal osseous and foci, furthermore the variance had statistical significance (P<0.01)。The non-sclerotic groupThe concentration level of three drugs and their metabolite in foci were much lower than that of A and B segment subnormal osseous and the variance had statistical significance (P<0.01), The concentration level of three drugs and their metabolite in A segment subnormal osseous were lower than that of B segment subnormal osseous (table 2) but the variance had not statistical significance (P>0.05)The Sclerotic groupThe concentration level of three drugs and their metabolite in A segment subnormal osseous were lower than that of B segment subnormal osseous but the variance had not statistical significance (P>0.05), The concentration level of three drugs and their metabolite in A segment subnormal osseous were lower than that of iliac tissue and the variance had statistical significance (P<0.01), in B segment subnormal osseous were similar to that of iliac tissue (table 3) and the variance had not statistical significance (P>0.05), Conclusion.1. the concentration level of antituberculosis drugs in foucs is below to minimal inhibitory concentration (MICs) of them.2. the concentration level of antituberculosis drugs in A segment subnormal osseous is low which inside the range of 4mm focus fringe in sclerotic group, when surgery this fraction could be resected partially. the concentration level of antituberculosis drugs in A segment subnormal osseous is high which inside the range of 4mm focus fringe in non-sclerotic group. when surgery this fraction could be retained partially.3. the concentration level of antituberculosis drugs in sclerotic wall is greatly below to minimal inhibitory concentration (MICs) of them, when surgery this fraction should be resected thoroughly. |
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