介绍一种改良的麦康凯培养基

用麦康凯（ＭＣＡ）培基粉作基础,加入６苯甲酰萘基磷酸二钠盐改良ＭＣＡ培养基。经多种肠道菌检测证实本法可同时观察乳糖和磷酸酶反应,在原始平板上根据菌落的颜色,直接区分肠杆菌科和非发酵菌之菌落,简化了鉴定手续,节约了鉴定时间,对进一步选择鉴定板有很大帮助。     

High Bilirubin Levels Interfere with Serum Tartrate-Resistant Acid Phosphatase Determination: Relevance as a Marker of Bone Resorption in Jaundiced Patients

Serum tartrate-resistant acid phosphatase (TRAcP) activity is considered to be a biochemical marker of bone resorption. Recently, a lack of specificity of collagen-related markers for assessing bone turnover has been observed in patients with chronic liver disease. Thus, it could be of great interest to determine serum TRAcP activity in such patients. However, nonspecificity of the analytical reaction could occur when hemolyzed, lipemic, or icteric specimens are analyzed. Therefore, we have studied the interference caused by bilirubin in the measurement of serum TRAcP activity using the Hillmann method. The interference was assessed in two pools of serum containing different bilirubin concentrations but with similar total AcP levels. Mixing proportional parts of the two pools, 10 samples were also obtained. Serum activities of total AcP and TRAcP, and the concentration of bilirubin were measured in the 10 samples. Both the actual and the expected values obtained by theoretical calculations were compared. Serum bilirubin values of 2.4 mg/dl showed a negative interference of 15% in the determination of serum TRAcP activity, whereas values of bilirubin higher than 10 mg/dl interfered totally with the measurement of serum TRAcP. Bilirubin did not interfere with the total AcP determination. This study clearly shows the interference of bilirubin in the determination of serum TRAcP. This finding should be considered when bone metabolism disorders are evaluated in jaundiced patients. Received: 6 April 1998 / Accepted: 1 October 1998     

某些酶活性变化对评价生长发育期大鼠锌营养的意义

雄性断乳大鼠随机分为低锌组、高锌组、正常锌组和低锌组的高锌饲料对饲组。喂相应饲料２０天后,低锌组的部分大鼠改喂高锌饲料（低 高锌组）,高锌组的部分大鼠改喂低锌饲料（高 低锌组）。分别于实验期０、２０、５０和７０天时处死。测定血浆碱性磷酸酶、５′ 核苷酸酶和铜 锌型超氧化物歧化酶活性、血浆锌及肾脏锌含量变化,以考核它们对评价锌营养状况的意义。结果表明：碱性磷酸酶、５′ 核苷酸酶以及血浆锌浓度变化对缺锌或／和补锌反应敏感。短期缺锌时（２０天）碱性磷酸酶活性就显著降低,并随缺锌时间延长该酶活性持续降低,而短期补锌（３０天）后又能显著增加其活性。受锌营养状况影响,血浆５′ 核苷酸酶活性也有类似的变化趋势。但这些酶活性又受增龄因素的影响,即随实验期增加,正常锌组碱性磷酸酶活性呈降低趋势,而５′ 核苷酸酶活性则呈增加趋势。长期严重缺锌可以影响铜 锌型超氧化物歧化酶活性;补锌后在实验期内未见到它的活性显著增加。随实验期增加,低锌组血浆锌持续降低,５０天时,显著低于高锌组;７０天时,低锌组和高 低锌组的血浆锌也显著低于高锌和低 高锌组。这些结果提示,碱性磷酸酶和５′ 核苷酸酶的活性及血浆锌变化对评价大鼠锌营养状况有一定的意?     

Purification of alkaline phosphatase from extracellular vesicles of fracture callus cartilage

Summary Extracellular matrix vesicles from fracture callus cartilage were isolated by differential centrifugation and resolved by equilibrium centrifugation on a discontinuous sucrose gradient into two bands. The phosphohydrolytic activity towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was distributed similarly after differential and equilibrium centrifugation suggesting the association of this activity with the matrix vesicles. The two bands isolated by equilibrium centrifugation of the partially purified vesicular preparation demonstrated high levels of alkaline phosphatase activity. Observed with an electron microscope, the 1.07–1.14 g/cm³ band from the gradient was enriched in electron luscent matrix vesicles while the 1.27 g/cm³ band contained electron dense matrix vesicles. Enzymatic analysis of the 1.27 g/cm³ band indicated a slight contamination due to the presence of mitochondria and lysosomes while the 1.07–1.14 g/cm³ band gave no enzymatic indication of subcellular contamination. A phosphohydrolytic enzyme active towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was purified from the 1.07–1.14 g/cm³ fraction by DEAE-cellulose column chromatography. Electron micrographs of callus cartilage sections demonstrated densification of the plasma membrane and matrix vesicles following substrate incubation with-glycerophosphate or tetrasodium pyrophosphate. The histochemical and biochemical data indicate that a phosphatase, with multiple substrate specificity, is a component of fracture callus cartilage matrix vesicles.     

Methoxyethylmercury nephrotoxicity: effects on enzymuria and kidney function

The fungicide, methoxyethylmercury chloride, was given in a saline solution to four groups of Sprague-Dawley C D rats (5 , 5 ) as a single injection (IP) of 0, 0.5, 1.0, and 2.0 mg Hg/kg. In a three-day period, no changes were observed in urine collected every 24 h from rats given 0 or 0.5 mg Hg/kg; 1 mg Hg/kg induced only a transient increase of urine gamma glutamyl transferase (x 4) and alkaline phosphatase (x 2.5) on the day 2; 2.0 mg Hg/kg caused an early increase of enzymuria (day 1 and day 2) and a decrease of Na⁺, Cl^–, K⁺, urea, and creatinin excretion. Urine enzymes and total mercury excretion were higher in males. These time-related variations of enzymuria, compared to previous results with Hg Cl₂, could reflect the existence of metabolites more toxic than the native compound.     

Endosteal surfaces in hyperparathyroidism: An enzyme cytochemical study on low-temperature-processed,glycol-methacrylate-embedded bone biopsies

Summary Alkaline phosphatase (AIP) and tartrate-resistant acid phosphatase (TRAP) activities have been studied comparatively in needle biopsies of the iliac crest of four cases of secondary hyperparathyroidism (renal osteodystrophy). AIP activity was associated with the plasma membrane of osteoblasts and their processes, of reticular cells of bone marrow and of young osteocytes of osteoid borders and woven bone. Moreover, it was detected in the fibroblast-like cells of the endosteal fibrosis. These cells were orderly in arrangement and were parallel to the endosteal surfaces near zones of bone formation. They were disorderly near zones of bone resorption. A strong TRAP-positive reaction was present in osteoclasts and mononuclear cells of endosteal fibrosis and in osteocytes located near active osteoclasts and in woven bone. These results suggest that the socalled fibrosis of hyperparathyroidism, rather than representing reparative, inert tissue, consists of osteoblastlike cells, probably precursors of osteoblasts derived by parathormone-stimulated proliferation of AIP-positive stromal cells of bone marrow, and of TRAP-positive, mononuclear cells, probably preosteoclasts. Moreover, they show that TRAP activity can be present in osteocytes, probably under stimulation by the same factors which stimulate osteoclast activity. The histochemical demonstration of AIP and TRAP facilitates the morphological diagnosis of metabolic bone disease and may improve knowledge of bone physiopathology.     

Kinetics of β-glycerophosphate-induced endochondral mineralization In vitro. Calcium accumulation,alkaline phosphatase activity,and effects of levamisole

Summary Isolated mesenchymal limb bud cells from day-12 mouse embryos grown at high density in organoid culture at the medium/air interphase differentiate into chondrocytes and form cartilage nodules. Upon addition of -glycerophosphate (-GP), cartilage undergoes endochondral mineralization. This -GP-induced mineralization was investigated by measuring the calcium content in the cultures and the activity of alkaline phosphatase (AP) in the cell mass and the medium. Calcium incorporation depended on the amount of -GP added. After continuous treatment, mineralization began on day 8 of the culture period and increased linearly until day 15. In long-term cultures, periodical treatment for 6 days caused an increase in mineralization the older the cultures were, but the slope of increase was proportionately less steep. Treatment at the latest period on days 19–24 resulted in a markedly reduced mineralization. After short-term treatment (48 hours), mineralization increased also the older the cultures were and proceeded during further cultivation in -GP-free medium. This kinetic behavior indicates a dependency of mineralization on cartilage maturation in this in vitro system. AP activity increased enormously and nearly logarithmically in the cell mass in -GP-free medium, whereas -GP treatment inhibited this drastic increase. In the medium, considerable activities of AP were also measurable from day 10 onward. It increased in -GP-free medium up to day 14, but was diminished after mineralization had been induced. Levamisole inhibited AP activity dose dependently when added directly to the enzyme-containing medium (100% inhibition at 10^-3 M). Added to the cultures from day 7 to 14, it partially inhibited AP activity and mineralization at 5×10^-5 M; mineralization was totally inhibited at 10^-3 M, but AP activity was still present. This high concentration was cytotoxic, as revealed ultrastructurally and by GAG estimation. This in vitro system comprises cartilage development and maturation, -GP-inducible endochondral mineralization, and final degenerative changes; it may be an appropriate model for investigations on endochondral mineralization.     

Alkaline phosphatase and peptidase levels in invertebrate cartilage

Summary Cartilage is encountered in the skeletons of many advanced invertebrates, yet it never calcifies or is replaced by bone. In an attempt to account for the absence of bone in invertebrates, we tested a hypothesis proposing that absence or inadequate quantities of several enzymes associated with vertebrate osteogenesis may underlie the failure of the invertebrates to evolve bone. The enzymes examined were alkaline phosphatase, alanyl -naphthylamidase, and neutral protease. Their activities were measured in the gill cartilage of the Atlantic horseshoe crab, Limulus polyphemus, and the odontophore cartilage of the marine whelk, Busycon canaliculatum. Animals were collected from the Cape Cod area. Samples of cartilage of Limulus perichondrium, various nonskeletal tissues, and neonatal rat calvaria, the latter as a reference standard, were homogenized in 0.1 M phosphate buffer (pH 7.1) and analyzed for protein content and the above-mentioned enzyme activities. Alkaline phosphatase specific activity was readily detected in most tissues except the invertebrate cartilage specimens in which it was present only at near-trace levels. Naphthylamidase and protease activities were present in all tissues. In a single experiment, higher phosphatase values were recorded for Limulus cartilage retaining perichondrium, but in a subsequent trial assaying cartilage retaining perichondrium, denuded cartilage, and isolated perichondrium separately, it was demonstrated that phosphatase activity resided primarily within the perichondrium. Exposure of thick cryostat sections to p-nitrophenyl phosphate confirmed the suspicion that alkaline phosphatase activity was present principally in the perichondrium. In view of the strong association between alkaline phosphatase and vertebrate cartilage mineralization it is proposed that extremely low levels of this enzyme may contribute to the failure of invertebrate cartilage to be replaced by bone.     

Ipriflavone inhibits osteoclast differentiation in parathyroid transplanted parietal bone of rats

Summary Ipriflavone, a synthetic isoflavone-derived flavonoid, was shown to have inhibitory effect on bone resorption. In order to study its mechanism of action directly on bone, 46 female Wistar rats were divided into six groups and medicated orally for 25 days as follows: groups 1 and 2 were given 1% carboxymethylcellulose solution (vehicle), groups 3, 4, 5, and 6 were administered ipriflavone at doses of 0.178, 0.356, 0.712, and 1.424 mmol/kg/day (suspended in vehicle), respectively. On the 22nd day, parathyroid glands, taken from donor rats, were transplanted in contact with the outer surface of the periosteum of both the right and the left parietal bones of rats from groups 2, 3, 4, 5, and 6. The group 1 rats underwent sham operation. Bone histomorphometry, performed on the ectocranial periosteum of parietal bones, showed that absolute erosion boundary, absolute eroded area, absolute erosion depth, number of tartrate-resistant acid phosphatase (TRAP)-positive polinucleated osteoclasts, and number of TRAP-positive mononucleated cells decreased in ipriflavone-treated rats compared with group 2 rats. The reduction was roughly proportional to the increase of drug dosage and reached statistical significance in rats of groups 5 and 6. The same parameters were extremely low in group 1 rats. Mineral apposition rate did not differ in any of the groups. Significant increase of serum calcium and significant decrease of serum phosphate were found in group 2 rats compared with group 1 rats, whereas no differences from controls were detected in ipriflavone-treated animals.The results demonstrate that ipriflavone has a direct inhibitory effect upon bone resorption, probably by reducing recruitment or differentiation of osteoclasts, rather than by inhibiting the resorption activity of differentiated osteoclasts. Ipriflavone also seems to exert a protective action against parathyroid hormone (PTH) diffusion from the site of parathyroid gland transplantation.     

Creatine kinase isoenzyme (CK-BB) in combination to prostatic acid phosphatase measured by RIA in the diagnosis of prostatic cancer

Summary Creatine kinase isoenzyme (CK-BB) measured by mass was used to determine its value in the early diagnosis of prostatic cancer. Sera of patients with prostatic carcinoma of various stages (treated and untreated) were compared to normal male sera and sera of patients with benign hyperplasia of the prostate (BPH) with respect to CK-BB. The sera were simultaneously tested for PAP content. The sensitivity of the CK-BB-RIA was 1.63+/-0.08 g/l and reproducibility in the higher and lower concentration range 7.6% and 10.5%, respectively. CK-BB alone or in combination with PAP is no marker for early detection of prostatic cancer. In individual cases changes occurred similar to those found with a malignant growth of the prostate.