[Study on changes of coagulation state, microthrombus and vascular density in the model of iron accumulation osteoporosis].

OBJECTIVE: To understand changes of coagulation state, microthrombus, microvascular bed and bone density in the osteoporosis model of iron accumulation, and explore the influence of iron accumulation in aspects of osteoporosis on coagulation function and blood vessels. METHODS: Tewnty-four male SPF SD rats aged 6 months were selected, which with the average body weight (250±20) g, which were divided into control group and iron accumulation group according to random number table, 12 rats in each group. Iron accumulation group was intervened by intraperitoneal injection of ferric ammonium citrate 90 mg / kg, and control group was intraperitoneally injected with equal volume of normal saline, twice a week for 9 weeks. After intervention, serum ferritin, coagulation function, microthrombus, vascular density, and three-dimensional morphological reconstruction and spatial structure parameters of the distal femur trabeculae were measured and statistically analyzed. RESULTS: Serum ferritin of iron accumulation group (136.36±35.41) µg / L was higher than control group (68.44±16.86) µg / L(P<0.05). Bone mineral density (BMD) of iron accumulation group (0.167±0.024) g / cm3 was lower than control group(0.400±0.030)g / cm3. Fibrinogen of iron accumulation group (2.03±0.13) g / L was increased than that of control group (1.78±0.46) g / L, D-dimer contents of iron accumulation group (534.95±31.81) ng /ml was increased than that of control group (329.02±84.99) ng /ml, while thrombin time (39.64±2.18) s and prothrombin time(8.70±0.39) s of iron accumulation group were shorter than that of control group (44.92±2.98) s, (9.44±0.49) s (P<0.05). After ink staining, microvessel density in iron accumulation group (17.46±2.07)% was significantly reducedcompared with that of control group(23.81±2.98)%(P<0.05). HE and MSB staining which showed microthrombus in bone marrow of iron accumulation rats, as well as microthrombus in myocardium. CONCLUSION: In the osteoporosis model with the influence of iron accumulation, iron accumulation had a significant influence on the coagulation function, and the blood was relatively hypercoagulable. The bone vascular bed uas reduced, and there were microthrombus in the bone marrow. Hypercoagulable state of blood and formation of microthrombi may be important factors influencing the occurrence of iron accumulation osteoporosis.

Bone Microthrombus Promotes Bone Loss in Iron Accumulation Rats.

In the present study, we investigated the changes of the coagulation state, bone microthrombus, microvascular bed and bone density levels in iron accumulation rats. Meanwhile,the effect of anticoagulation therapy on bone mineral density was further investigated. We established two groups: a control (Ctrl) group and an iron intervention (FAC) group. Changes in coagulation function, peripheral blood cell counts, bone microthrombus, bone vessels and bone mineral density were compared between the two groups. We designed the non-treatment group and treatment group to study the changes of bone mineral density by preventing microthrombus formation with the anticoagulant fondaparinux. We found that the fibrinogen and D-dimer contents were significantly higher, whereas the thrombin time (TT) and prothrombin time (PT) were significantly shorter in the FAC group. After ink staining, the microvascular bed in the FAC group was significantly reduced compared with that in the Ctrl group. HE and Martius Scarlet Blue (MSB) staining showed microthrombus in the bone marrow of the iron accumulation rats. Following anticoagulation therapy, the bone microcirculation vascular bed areas in the treatment group rats were significantly increased. Furthermore, the bone mineral density was increased in the treatment group compared with that in the non-treatment group. Through experiments, we found that the blood in iron accumulation rat was relatively hypercoagulable; moreover, there was microthrombus in the bone marrow, and the bone vascular bed was reduced. Additionally, anticoagulation was helpful for improving bone microcirculation, reducing microthrombus and decreasing bone loss.

Oxygen uptake rate optimization with nitrogen regulation for erythromycin production and scale-up from 50 L to 372 m3 scale.

Effects of different nitrogen sources on the erythromycin production were investigated in 50 l fermenter with multi-parameter monitoring system firstly. With the increase of soybean flour concentration from 27 g/l to 37 g/l to the culture medium, the erythromycin production had no obvious increase. Whereas adding corn steep liquor 15 g/l in the medium was beneficial for the production of erythromycin, the maximum erythromycin production was 22.2% higher than that of the control. It was found that corn steep liquor can regulate and enhance the oxygen uptake rate (OUR) which characterizes the activity of the microbial metabolism by inter-scale observation and data association. Both Intracellular and extracellular organic acids of central metabolism were analyzed, and it was found that the whole levels of lactic acid, pyruvic acid, citric acid, and propionic acid were higher than those of control before 64th h. The consumption amount of amino acids, which could be transformed into the precursors for erythromycin synthesis (i.e. threonine, serine, alanine, glycine and phenylalanine), were elevated compared with the control in erythromycin biosynthesis phase. The results indicated that corn steep liquor can regulate OUR to certain level in the early phase of fermentation, and enhance the metabolic flux of erythromycin biosynthesis. Erythromycin production was successfully scaled up from a laboratory scale (50 l fermenter) to an industrial scale (132 m(3) and 372 m(3)) using OUR as the scale-up parameter. Erythromycin production on industrial scale was similar to that at laboratory scale.

Enhancement of erythromycin A production with feeding available nitrogen sources in erythromycin biosynthesis phase.

Effects of feeding different available nitrogen sources from 80 h in erythromycin biosynthesis phase on the erythromycin A (Er-A) production were investigated in 50 l fermenter. Feeding corn steep liquor and yeast extract, the Er-A production was enhanced, while the biotransformation from erythromycin C (Er-C) to Er-A had no increase. When ammonium sulphate was fed at high feeding rate, the maximal Er-A production and ratio of Er-A to Er-C were 7953 U/ml and 98.18:1 at 184 h, respectively, which were higher than that of the control (6742 U/ml and 5.47:1). The feeding ammonium sulphate process was successfully scaled up from 50 l to 25 m(3) fermenter. The maximal Er-A production reached 7938 U/ml at 203 h, which was enhanced by 22.1% compared with the control (6501 U/ml at 192 h). The ratio of Er-A to Er-C was 24.05:1, which was higher than that of the control (4.77:1).

Application of oxygen uptake rate and response surface methodology for erythromycin production by Saccharopolyspora erythraea.

A process for efficient production of erythromycin by Saccharopolyspora erythraea using statistical designs and feeding strategy was developed. The critical nutrient components were selected in accordance with fractional factorial design and were further optimized via response surface methodology. Three significant components (ZnSO(4), citric acid threonine) were identified for the optimization study. The optimum levels of these significant variables were determined with Box-Behnken design, which were ZnSO(4) 0.039 g/l, citric acid 0.24 g/l and threonine 0.42 g/l, respectively. A novel feeding strategy based on oxygen uptake rate (OUR) measurement was developed successfully to increase the flux of erythromycin biosynthesis, in which the optimized nutrient components was fed in the 50 l stirred bioreactor when OUR began to decline at 46 h. The maximum erythromycin production reached 10,622 U/ml, which was 11.7% higher than the control in the same cultivation conditions. It was the first report to integrate physiological parameter OUR and statistical methods to optimize erythromycin production.

[Quantitative studies of the production phase in rHSA fermentation].

The model equations of the production phase of rHSA fermentation were derived on the base of both elemental balance and metabolic balance, then the unknown parameters of the model were estimated by multivariable optimization. The possible reasons of discrepancy of production rate between different period of fermentation were discussed. The model could preferably described the relations between different macroscopic reaction rates of the process and keys for the high-efficiency expression of HAS were deduced.