Incidence of breakage of human RBCs frozen with 40-percent wt/vol glycerol using two different methods for storage at -80 degrees C

BACKGROUND: We reported previously that the incidence of breakage was 34.2 percent when human RBCs were frozen with 40-percent wt/vol glycerol in polyolefin plastic bags stored in aluminum containers at -80 degrees C and subjected to transportation. When human RBCs were frozen with 40-percent wt/vol glycerol at -80 degrees C in PVC plastic bags placed in polyester plastic bags and stored in rigid corrugated cardboard boxes, transportation resulted in a 2.4-percent incidence of breakage. The present study was done to confirm this incidence of breakage. STUDY DESIGN AND METHODS: The Meryman- Hornblower freezing method was compared to the Naval Blood Research Laboratory (NBRL) method of freezing for incidence of bag breakage. Human RBCs frozen by the Meryman-Hornblower method with 40-percent wt/vol glycerol with supernatant glycerol and stored in polyolefin plastic bags in aluminum containers at -80 degrees C were stored at the NBRL from 1974 to 2002. With the NBRL method, human RBCs frozen at -80 degrees C without supernatant glycerol in the 800-mL PVC plastic primary bag inside a polyester plastic bag in a rigid corrugated cardboard box were stored at the NBRL from 1984 to 2002. RESULTS: The incidence of breakage for 532 units of RBCs that had been frozen by the Meryman- Hornblower method and stored in aluminum containers was 47.3 percent for nontransported units. RBCs that had been frozen by the NBRL method and stored in rigid corrugated cardboard boxes exhibited breakage of 2.4 percent for 2424 nontransported units and 6.7 percent for 633 transported units. DISCUSSION: The incidence of breakage was significantly lower for RBCs frozen by the NBRL method than for the RBCs frozen by the Meryman-Hornblower method.     

The in vitro quality of red blood cells frozen with 40 percent (wt/vol) glycerol at -80 degrees C for 14 years, deglycerolized with the Haemonetics ACP 215, and stored at 4 degrees C in additive solution-1 or additive solution-3 for up to 3 weeks

BACKGROUND: Red blood cells (RBCs) frozen with 40 percent (wt/vol) glycerol, stored at -80 degrees C (mean temperature; range, -65 to -90 degrees C) for 14 years, deglycerolized in the Haemonetics automated cell processor (ACP) 215 with the 325-mL disposable bowl, and stored at 4 degrees C in additive solution (AS)-1 or AS-3 for 21 days were evaluated. STUDY DESIGN AND METHODS: A total of 106 units of citrate phosphate dextrose adenine-1 RBCs were frozen with 40 percent (wt/vol) glycerol in the original 800-mL polyvinylchloride plastic bag and stored in corrugated cardboard boxes at -80 degrees C for 14 years. The thawed units were deglycerolized with the ACP 215 with a 325-mL disposable bowl and stored in AS-1 or AS-3 at 4 degrees C for 21 days. RESULTS: The freeze-thaw recovery value was 94 +/- 4 percent (mean +/- SD), the freeze-thaw-wash recovery value was 80 +/- 7 percent, and there was no breakage. Thirty-eight units were processed as 19 pairs. Two units of ABO-matched units were thawed, pooled, divided equally into two units, and deglycerolized. One unit was stored in AS-1 and the other in AS-3 at 4 degrees C for 21 days. Units stored in AS-1 exhibited significantly greater hemolysis than those stored in AS-3. CONCLUSIONS: Acceptable results were achieved when RBCs frozen at -80 degrees C for 14 years were deglycerolized in the ACP 215. Deglycerolized RBCs in AS-1 exhibited significantly higher hemolysis than those in AS-3 after storage at 4 degrees C for 7 to 21 days.     

The safety and therapeutic effectiveness of human red cells stored at - 80 degrees C for as long as 21 years

Human red cells frozen by various methods have been stored in the frozen state at -80 degrees C for as long as 21 years. This report discusses: red cells frozen with 42 percent weight per volume (wt/vol) glycerol in an ionic medium in a polyvinylchloride (PVC) plastic bag using the Cohn method; red cells frozen with 45 percent wt/vol glycerol in a low ionic medium in a PVC plastic bag using the Huggins method; red cells frozen with 40 percent wt/vol glycerol in an ionic medium in a polyolefin plastic bag using the Meryman-Hornblower method; and red cells frozen with 40 percent wt/vol glycerol in an ionic medium in a standard 600-ml or an elongated 800-ml PVC plastic primary collection bag with an adapter port using the Naval Blood Research Laboratory (NBRL) method. After frozen storage for as long as 21 years by the four methods described above, the thawed red cells were deglycerolized with 50 to 150 ml of 12 percent sodium chloride and 1.5 to 2.0 l of sodium chloride-glucose or sodium chloride-glucose-phosphate solution. After washing and storage at 4 degrees C for 24 hour, the red cells had a mean freeze-thaw-wash recovery value of 90 percent, a mean 24-hour posttransfusion survival value of 85 percent, a mean index of therapeutic effectiveness of 75 percent, normal or slightly impaired oxygen transport function, and minimal hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo measurements of human RBCs frozen with glycerol and subjected to various storage temperatures before deglycerolization and storage at 4 degrees C for 3 days

BACKGROUND: This study was designed to assess the effects of changes in storage temperature of frozen RBCs such as might occur during a malfunction of the -80 degrees C mechanical freezer or during shipment. STUDY DESIGN AND METHODS: Fifteen participants donated blood for autologous transfusion of RBCs; all RBCs were frozen with 40-percent (wt/vol) glycerol. Five subjects received RBCs that were stored at -80 degrees C alone before transfusion. Five subjects received RBCs that were stored initially at -80 degrees C, then at -40 degrees C for 4 weeks, and finally at -80 degrees C before transfusion. Five subjects received RBCs that were stored at -80 degrees C, then at -20 degrees C for 2 weeks, and finally at -80 degrees C before transfusion. After deglycerolization, the RBCs were stored at 4 degrees C in a sodium chloride-glucose solution for 3 days before transfusion. RESULTS: No significant differences were observed in freeze-thaw recovery, freeze-thaw-wash recovery, 24-hour posttransfusion survival, index of therapeutic effectiveness, or RBC ATP levels. Greater hemolysis and reduced RBC K+ levels were observed in the units stored at -80 degrees C/-40 degrees C/-80 degrees C and in those stored at -80 degrees C/ -20 degrees C/-80 degrees C compared with the units stored at -80 degrees C alone, but these differences did not affect the 24-hour posttransfusion survival. CONCLUSIONS: The results of this study indicated that RBCs frozen with 40-percent (wt/vol) glycerol can be stored at -40 degrees C for 4 weeks or at -20 degrees C for 2 weeks between periods of frozen storage at -80 degrees C with satisfactory results.     

种植牙用钛合金Ti-30Nb-8Zr-2Mo硬度及弹性模量

背景:目前广泛应用于临床的纯钛和Ti-6AI-4V种植体材料中存在着铝和钒的潜在毒性及弹性模量太大易造成界面应力屏障等问题.目的:对自行研制的新型钛合金Ti-30Nb-8Zr-2Mo进行硬度及弹性模量性能测试.设计、时间及地点:观察实验,于2003-03/2006-02在河北工业大学材料学实验室完成.材料:钛合金制备用原材料海绵钛纯度≥99%、钼粉纯度≥99%、铌条≥99.9%、海绵锆≥99.4%.方法:在均匀化退火、热煅、固溶后对试样显微硬度进行测量,对时效后的样品进行压缩试验.主要观察指标:硬度值和应力应变曲线.结果:研究显示800℃0.5 h固溶后得到最大程度的强化合金,固熔合金硬度变化不大,时效后硬度显著增强,试样的抗压强度为1 054 MPa,压缩弹性模量达到16.5 GPa.结论: 自行研制的新型钛合金Ti 30Nb-8Zr-2Mo硬度和弹性模量均达到种植体材料的性能要求.     

Prorenin-renin conversion by the contact activation system in human plasma: role of plasma protease inhibitors

Acid-pretreated normal human plasma generates renin activity at 0 degree C and neutral pH by the activation of prorenin. The activation is caused by kallikrein generated from prekallikrein by activated factor XII. Nonacidified plasma also generates renin at 0 degree C, but at a lower rate (cold-promoted activation). In normal plasma, 14% +/- 1% of prorenin (mean +/- SEM, n = 30) was activated during incubation at 0 degree C for 7 days (range 6% to 26%). Cold-promoted activation of prorenin was within the normal range in plasma deficient in factor XI, X, IX, VIIIC, VII, V, prothrombin, or high mol wt kininogen. Cold-promoted activation of prorenin was less than or equal to 1% in plasma deficient in factor XII or prekallikrein. Reconstitution of these plasmas with highly purified factor XII or prekallikrein restored normal prorenin activation. Correction of high mol wt kininogen deficiency had no effect. Thus cold-promoted activation of prorenin depends on the presence of factor XII and prekallikrein, whereas the other clotting factors are not essential. The influence of the inhibitors C1 esterase-inhibitor, alpha 2-macroglobulin, antithrombin III, and alpha 1-antitrypsin on the activation of prorenin was studied in factor XII-deficient plasma from which one or more of these inhibitors had been selectively removed by immunoadsorption. Factor XII was subsequently added, and the generation of renin at 37 degrees C was observed after complete factor XII-high mol wt kininogen-mediated activation of prekallikrein induced by dextran sulfate. No activation of prorenin was observed at 37 degrees C after depletion of C1 esterase inhibitor, alpha 2-macroglobulin, antithrombin III, or alpha 1-antitrypsin. When prekallikrein was activated in plasma depleted of both C1 esterase-inhibitor and alpha 2-macroglobulin, 6% of prorenin was activated in 2 hours at 37 degrees C. After additional depletion of antithrombin III, the activation increased to 47%. These results indicate that the contact activation system is capable of activating prorenin in plasma at physiologic pH and temperature when the three most important kallikrein inhibitors, C1 esterase-inhibitor, alpha 2-macroglobulin, and antithrombin III, are absent.     

STABILITY OF STEROID OINTMENTS DILUTED WITH COMPOUND ZINC PASTE B.P.

The chemical stability of a range of corticosteroid ointments diluted with Compound Zinc Paste B.P. was studied by high-performance liquid chromatography (HPLC). Betamethasone dipropionate degraded by first-order kinetics at both 25 degrees C and 32 degrees C. At 25 degrees C the mean first-order reaction rate constant was 9.58 x 10(-3)/day, with a t90 (time to reach 90% of the original concentration) of 11.0 days. At 32 degrees C the mean first-order reaction rate constant was 2.42 x 10(-2)/day, with a t90 of 4.4 days. Of the other steroids studied, Fluocinolone acetonide was the least stable, with 33.8% remaining after a 7-day storage period at 25 degrees C. Betamethasone-17-valerate and fluocinonide were of intermediate stability between betamethasone dipropionate and fluocinolone acetonide. None of the steroid ointments studied had adequate stability in Compound Zinc Paste B.P. to allow extemporaneous dilution with this base.     

Influence of body temperature on indinavir crystallization under loop of Henle conditions

OBJECTIVES: Indinavir is a protease inhibitor used in the therapy of HIV-1+ patients. It causes indinavir stone formation. It has been shown to precipitate in the loop of Henle (LH) at plasma concentrations (conc[P]) of approximately 8 mg/L. Those experiments were performed at room temperature. Given the influence of temperature on crystallization in general, and solubility of indinavir in particular, we repeated the experiments under physiological (body) temperature conditions. METHODS: Test solutions contained indinavir concentrations of 100-750 mg/L at ionic strengths varying from 0 to 800 mM simulating conditions in the proximal tubule and the LH. Solutions were titrated with base (NaOH) to find the pH value where nucleation is initiated. Experiments were conducted at room temperature (20 degrees C) and repeated under constantly monitored (body) temperature (37 degrees C). RESULTS: Experiments at 20 degrees C confirmed our previous results. At 37 degrees C, the relationship between pH and indinavir concentration remained inversely proportional. Again, the LH was confirmed as the most likely localization of crystallization. However, at 37 degrees C precipitation occurred at a lower urinary concentration (100 versus 125 mg/L) and within a lower pH range (6.67-7.26 versus 7.23-7.44). This lower urinary concentration corresponds to a lower conc[P] [critical value (CV)] of 6.41 mg/L, as compared with 8.01 mg/L at 20 degrees C. CONCLUSIONS: The CV is even lower at 37 degrees C than previously assumed. Plasma peak concentration above the CV of 6.4 mg/L will induce crystallization in the LH and should be avoided.     

Identification and characterization of specific binding proteins for growth hormone in normal human sera.

The well-recognized "big" forms (45,000-100,000 mol wt) of immunoreactive human growth hormone (hGH) in human serum have been reported to be random aggregates or formal polymers. However, we have now investigated the possibility that they are protein-bound forms. After incubation of monomeric 125I-hGH with normal serum, gel chromatography indicated a peak of bound 125I-hGH (at approximately 120,000 mol wt), which was completely displaced by excess unlabeled hGH. When serum alone was chromatographed two peaks of specific binding were subsequently detected, the major peak, eluting between 74,000 and 85,000 mol wt corresponded to the 125I-hGH-binding protein complex observed at approximately 120,000 mol wt. Using a mini-gel filtration system for separating bound from free hormone, binding of 125I-hGH by normal human serum was dependent on time (equilibrium was reached in 2 h at 21 degrees C), temperature (21 degrees C greater than 37 degrees C), Ca2+ and serum concentrations. Binding was reversible and highly specific for hGH, not being displayed by GH or prolactins from several species. Scatchard analysis revealed linear plots with an affinity (KA) of 0.32 +/- 0.06 X 10(9) M-1 (n = 7). Human serum with low endogenous hGH levels, when added to rabbit liver membranes, decreased the binding of 125I-hGH in this tissue in a dose-dependent manner. These data indicate that human sera contain a specific, high affinity binding protein for hGH and that this may account, at least in part, for the known size heterogeneity of GH in serum. Its effect on GH binding to target tissues may indicate a role for the binding protein in the regulation of GH action.     

Roles of liposome composition and temperature in distribution of amphotericin B in serum lipoproteins.

The role of liposome composition and temperature in the distribution of amphotericin B (AmB) with serum lipoproteins and the role of particle charge in AmB transfer to serum lipoproteins were determined. Serum obtained from healthy volunteers was incubated with known concentrations of AmB or different liposomal formulations of AmB (1 to 100 micrograms/ml) at 37 degrees C for various time intervals (5, 10, 20, 30, 45, and 60 min). After each interval, serum was removed and separated into high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions by an LDL-direct assay. The distribution of AmB (Fungizone) at 5 min through 1 h of incubation at 25 degrees C remained constant and was similar in the HDL and LDL fractions. At 37 degrees C, at 5 through 45 min of incubation, 54 to 61% of AmB was recovered in the HDL fraction; however, at 1 h more than 75% of the AmB concentration was recovered in the HDL fraction. In contrast, 87.5 to 92% AmB was recovered in the HDL fraction throughout the incubation when negatively charged liposomal AmB (dimyristoylphosphatidylcholine [DMPC]:dimyristoylphosphatidylglycerol [DMPG], 7:3 [wt/wt]) was used. With positively charged liposomes, 75 to 87.7% of AmB was recovered in the HDL fraction through the different time points studied. AmB incorporated into DMPC (neutral) and DMPG (negative) liposomes, and AmB was distributed in an HDL:LDL ratio of 6:4 following 1 h of incubation. Ninety percent of AmB and 80% of the lipid were found in the HDL fraction in a 3:1 molar DMPG:AmB ratio and in the LDL fraction in a 6:1 molar ratio. Lipid charge and temperature play a role in AmB distribution into serum lipoproteins. AmB and DMPG may contransfer as an intact drug-lipid complex to serum lipoproteins.     

Evaluation of red blood cells stored at -80 degrees C in excess of 10 years

BACKGROUND: RBCs frozen in 40 percent (wt/vol) glycerol are currently approved by the FDA and the AABB for storage at -80 degrees C for up to 10 years. STUDY DESIGN AND METHODS: This study examined 20 RBC units that had been cryopreserved in 40 percent (wt/vol) glycerol and stored at -80 degrees C for up to 22 years. Measures of the freeze-thaw-wash (FTW) recovery, ATP, 2,3-DPG, methemoglobin, RBC indices, morphology, and osmotic fragility were made immediately after deglycerolization and after 24 hours of storage at 4 degrees C. RESULTS: RBCs frozen for longer than 10 years had acceptable mean FTW recovery, normal oxygen transport function, RBC morphology, RBC indices, methemoglobin, and osmotic fragility. Statistical analysis indicated that the in-vitro viability and function of cryopreserved RBCs was not dependent on the length of frozen storage or postthaw storage at 4 degrees C but did correlate with the storage length at 4 degrees C before cryopreservation. CONCLUSION: The data reported in this study demonstrate that RBCs can be stored at -80 degrees C beyond 10 years with acceptable in-vitro quality and suggest that more defined criteria for the cryopreservation process be adopted.     

In vitro and in vivo measurements of gamma-radiated, frozen, glycerolized RBCs

BACKGROUND: Transfusion-associated GVHD results from the presence of viable lymphocytes in transfused allogeneic blood components. Viable immunocompetent lymphocytes have been detected in RBCs that were frozen with glycerol and washed before transfusion. STUDY DESIGN AND METHODS: The study reported here assessed the effect of irradiation on human RBCs frozen with 40-percent (wt/vol) glycerol and stored at -80 degrees C. In vitro and in vivo testing was done on human RBCs that were frozen with 40-percent (wt/vol) glycerol at -80 degrees C, with some units exposed to 2500 cGy of gamma radiation and others not irradiated, and that, after thawing and washing, were stored in a sodium chloride-glucose solution at 4 degrees C for 3 days before autologous transfusion. RESULTS: The glycerol-frozen RBCs treated with 2500 cGy before deglycerolization had a mean freeze-thaw-wash recovery of 87 percent and a mean 24-hour posttransfusion survival of 86 percent after storage for 3 days at 4 degrees C in a 0.9-percent NaCl and 0.2-percent glucose solution. For the nonirradiated units, the mean freeze-thaw-wash recovery was 85 percent and the mean 24-hour posttransfusion survival was 83 percent. CONCLUSION: These data show similar, acceptable results for RBCs frozen with 40-percent (wt/vol) glycerol at -80 degrees C and treated in the frozen state with 2500 cGy of gamma radiation and for RBCs that were not irradiated, all of which were washed and then stored in a sodium chloride-glucose solution for 3 days before autologous transfusion.     

构建自体骨碎末移植材料修复骨缺损的实验模型

学术背景:自体骨移植取材常需要开辟第二术区或在种植体周围取骨,额外增加创伤和感染机会,所以对局部小范围的骨质欠缺,可以考虑回收和利用预备种植体切除的自体骨末.目的:建立恢复种植体周围骨缺损的自体骨碎末骨移植材料的实验模型,观察材料与宿主的生物相容性反应.设计:单一样本观察.单位:大连医科大学口腔医学院.材料:实验于2005-08/2006-04在大连医科大学动物实验基地完成.实验动物为5只健康杂交家犬;种植体钛钉和Bio-Oss骨移植材料均由西安中邦钛生物材料有限公司设计制造并提供.方法:拔除家犬下颌第1,2,3前臼齿,3个月后行种植术.预备种植体窝,每只犬左右两侧各预备4个,共40个.在每个种植窝内,各植入种植体钛钉1枚,共40枚.用种植转孔时收集的自体骨碎末、Bio-Oss骨移植材料及两者1:1混合骨碎末恢复种植体颊侧单壁人为骨缺损,以未植骨作空白对照.主要观察指标: ①种植术后第9周时观察各组骨量的恢复情况、X射线片观察牙槽骨高度、骨小梁致密度及骨整合情况. ②应用亚甲基蓝-碱性品红法观察组织学变化.结果:5只家犬钛钉无脱落,均纳入结果分析. ①一般情况及骨缺损量:种植术后9周,创口愈合均良好,钛钉稳定,总存留率为100%.植入自体骨碎末的骨缺损量小于空白对照组(P＜0.01);植入混合骨碎末的平均骨缺损量最小,说明恢复最佳. ②骨量的恢复情况:X射线片显示40颗钛钉外周均与骨组织紧密接触,愈合良好. ③材料与宿主的组织相容性:低倍镜下见所有钛钉均被周围淡红色的致密骨组织紧密包绕,种植体与骨组织间无蓝色的软组织,产生了直接骨结合界面.结论:家犬建立自体骨碎末移植材料恢复种植体周围骨缺损的实验模型效果理想,材料与宿主间生物相容性好.     

Effect of cooling on the intracellular concentrations of Na+, K+ and Cl- in cultured human endothelial cells

Cooling is accepted as a practical way of lowering cell metabolism in vein grafts during coronary by-pass surgery. We have previously shown that low temperature causes endothelial cells to become detached, both in in vitro and in vein graft. In this study we have looked at the effect of cold on the concentrations of intra- and extracellular electrolytes. Human endothelial cells were grown on titanium grids for electron microscopy. The cells were incubated for 30 min at 37 degrees, 20 degrees, and 4 degrees C with cell culture medium containing human serum, and at 20 degrees and 4 degrees C with heparinized sodium acetate solution with serum, frequently used for flushing and distending vein grafts. Freeze-dried cells were then subjected to elemental X-ray microanalysis. The ambient fluid was analysed by flame photometry. At 20 degrees and 4 degrees C, intracellular concentration of sodium increased, and potassium decreased, compared with controls (37 degrees C). The changes in sodium concentrations were aggravated when cell culture medium was replaced by heparinized sodium acetate. The intracellular chloride concentration did not change when cells were stored in cold cell culture medium. The extracellular concentration of potassium increased with increasing incubation time at 4 degrees C. The connection between these findings and cell detachment is discussed.     

Thermal inactivation of L-thyroxin

We assessed the extent of inactivation of L-thyroxin induced by exposure to heat in the presence of two vehicles. Preparations of L-thyroxin in the dry powder form, or dispersed in the solvents propylene glycol (water-like) or ethoxylated castor oil (oil-like), were heated at temperatures ranging from 65 to 160 degrees C, for 5- to 15-min periods. Heating L-thyroxin to a temperature below that of cooked bovine ground meat (72 degrees C) produced less than 10% degradation. Thermal degradation was pronounced only above 90 degrees C, and was almost completed at 160 degrees C. L-Triiodothyronine was the only thermal degradation product identified after L-thyroxin was heated at 125 degrees C. In a separate experiment we measured the melting point of L-thyroxin, 148.81 degrees C. This value agrees closely with the observed thermal sensitivity. We conclude that L-thyroxin is not significantly degraded under conditions encountered during cooking of ground bovine meat for short times at moderate temperatures.     

Partial purification and characterization of contact activation cofactor.

The contact phase of intrinsic clotting involves Factor XI, Factor XII, Fletcher factor, and a fourth activity that we call contact activation cofactor (CAC). All four of these activities are reduced or absent in Dicalite-adsorbed plasma. A modified activated partial thromboplastin time assay for CAC has been defined by using a substrate of Dicalite-adsorbed plasma combined with partially purified sources of Factors XI and XII, and Fletcher factor. The following properties of CAC in plasma have been determined by using the assay: it is stable up to 60 min at 56 degrees C; gradually loses activity at 80 degrees C; is stable between pH 6 and 9; is precipitated by ammonium sulfate between 40% and 50% saturation; is slightly adsorbed by A1(OH)3; and is eluted from DEAE-cellulose after the major protein peaks. A purification procedure has been devised that separates CAC from other known clotting factors. Isolated CAC was less stable than CAC in plasma, but in the presence of dilute human serum albumin it retained full activity for 80 min at 56 degrees C. On gel filtration CAC had an apparent mol wt of 220,000 daltons. These properties are consistent with those described for Fitzgerald factor, which further supports the conclusion that CAC and Fitzgerald factor represent the same activity. Isolated CAC promoted the generation of activated Factor XI (XIa) in a mixture containing purified Factor XI, Factor XII, and kaolin. The amount of Factor XIa generated was proportional to the amount of added CAC. No time-consuming reaction between Factor XI or Factor XII and CAC could be demonstrated.     

FACTORS INFLUENCING THE PROTEIN BINDING OF BUMETANIDE USING AN EQUILIBRIUM DIALYSIS TECHNIQUE

Factors that influence the plasma protein binding of bumetanide were evaluated using equilibrium dialysis. It took approximately 12 h of incubation to reach an equilibrium between plasma and isotonic phosphate buffer of pH 7.4 containing 3% dextran using a Spectrapor 2 membrane (mol. wt cut-off = 12,000-14,000) in a water-bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. Bumetanide was fairly stable in both 4% human serum albumin (HSA) and in the isotonic phosphate buffer of pH 7.4 for up to 24 h. The binding of bumetanide to 4% HSA was constant (87.5 +/- 1.73%) at bumetanide concentrations ranging from 0.1 to 100 micrograms/ml. The extents of binding were 72.0, 83.3, 88.5, 90.2, 91.3 and 91.4% at albumin concentrations of 0.5, 1, 2, 3, 4 and 5 g/100 ml, respectively, and increased with a decrease in incubation temperature; the values bound were 94.6, 90.3 and 89.3% when incubated at 4, 22 and 37 degrees C, respectively. The binding of bumetanide was independent of the buffer composition used, the quantities of AAG (up to 0.32%), heparin (up to 40 units/ml), sodium azide (up to 0.5%) and anticoagulants (EDTA, heparin and citrate). The free fraction of bumetanide in rabbit plasma (2.91%) was significantly higher than in humans (1.98%) or rats (1.85%).     

The stability of immunological and biological activity of human thyrotropin in buffer: its temperature-dependent dissociation into subunits during freezing

The stability of thyroid-stimulating hormone (TSH) in buffer under various storage conditions was studied with regard to its immunoreactivity measured by an immunoradiometric assay and its bioactivity by a sensitive FRTL-5 cell bioassay. The immunoreactivity was well retained at 4 degrees C or 24 degrees C throughout the study period of 90 days. At -20 degrees C, however, it decreased proportionately with the storage time. The mean reduction was 42.1% at 90 days compared with that when stored at -80 degrees C. The bioactivity showed a similar course of change with its reduction of 44.3% at -20 degrees C in 90 days. The loss of both activities was attributed to the dissociation of human (h) TSH molecule into its subunits. The concentration of the alpha subunit of hTSH in those samples stored at -20 degrees C gradually increased from the initial undetectable level to that almost equivalent on a molar basis to the loss of immunoreactivity. The enrichment of albumin in the buffer to a level of more than 1.0% was effective in preventing the occurrence of such a phenomenon. These data indicate that hTSH, when frozen at -20 degrees C in buffer is gradually dissociated into its subunits, despite its outstanding stability for 90 days at both 4 degrees C and 24 degrees C. However, no apparent inconsistency between immunological and biological activities, was observed at any temperature between -20 degrees C and 24 degrees C.     

Effects of temperature, NaCl, and methicillin on penicillin-binding proteins, growth, peptidoglycan synthesis, and autolysis in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus strains produce a fifth penicillin-binding protein (PBP), PBP 2', with low affinity for beta-lactam antibiotics that is believed to represent a beta-lactam-insensitive peptidoglycan transpeptidase. In an effort to evaluate the adequacy of PBP 2' as an explanation of methicillin resistance, PBP 2' production and the responses of growth and peptidoglycan synthesis to methicillin under different environmental conditions have been compared. In the heterogeneous methicillin-resistant strain DU4916-K7, less PBP 2' was produced at 40 degrees C than at 30 degrees C, but inclusion of 5% (wt/vol) NaCl in the medium at 40 degrees C boosted PBP 2' production and allowed growth of the organism in the presence of 10 micrograms of methicillin per ml. When exponential-phase cultures were challenged with methicillin, growth and peptidoglycan synthesis were much more resistant at 30 degrees C than at 40 degrees C. Inclusion of NaCl in medium rendered growth and peptidoglycan synthesis more methicillin resistant at 40 degrees C. Hence, there was a good correlation between PBP 2' production and methicillin-resistant peptidoglycan synthesis under these conditions. However, PBP 2' production was increased by NaCl at 30 degrees C without markedly affecting the susceptibilities of growth and peptidoglycan synthesis to methicillin. Pregrowth of cells with methicillin, which was expected to boost PBP 2' production, seemed to increase the susceptibilities of growth and peptidoglycan synthesis to methicillin. Patterns of growth and peptidoglycan synthesis susceptibilities to methicillin which were similar to those described above were found in chloramphenicol-inhibited cultures, in which presumably no induction of PBP 2' could occur during the methicillin challenge period. Complex effects were noted in the combination of subinhibitory methicillin and NaCl. Growth of cells in the presence of NaCl stimulated their autolytic activity, which was further increased by growth with subinhibitory methicillin in addition to NaCl. It appears that NaCl enhances methicillin resistance by stimulating PBP 2' production and providing osmotic support but opposes it by stimulating autolytic activity which is exacerbated by the very low cross-linking of peptidoglycan in methicillin-resistant strains grown in the presence of methicillin.     

Enteral suspension of nifedipine for neonates. Part 2. Stability of an extemporaneously compounded nifedipine suspension

OBJECTIVE: To investigate the chemical, microbiological and physical stability of an extemporaneously prepared nifedipine oral suspension, packaged in disposable syringes and prepared in a hospital pharmacy for paediatric use. METHODS: Two different suspensions were prepared, from nifedipine tablets and drug powder, by using an autoclaved 1.0% solution of hypromellose as the vehicle. The final theoretical drug concentration was 1 mg/mL. Doses of 1.0 ml were packaged into a 2 ml syringe with a cap. Nifedipine suspensions were stored under three conditions: at room temperature (22 degrees C), in a refrigerator (6 degrees C) protected from light, and at room temperature (22 degrees C) exposed to artificial daylight (400 lux) under controlled circumstances. The nifedipine concentration was measured in suspensions protected from light on days 0, 1, 3, 5, 7, 14, 21 and 28, and in suspensions exposed to light at 0, 3, 6, 18 and 24 h, and on days 2, 3, 5 and 7 after preparation. Nifedipine was analysed by a reproducible and validated stability-indicating HPLC-method. Microbiological and physical stability of the nifedipine suspension samples protected from light were examined for 28 days. RESULTS: Mean nifedipine concentration remained over 90% of the initial concentration throughout the 4-week study period in light-protected unit-dose suspensions, prepared from either crushed tablets or drug powder with hypromellose 1.0%. When exposed to light, however, nifedipine decomposed rapidly. Photodegradation of nifedipine exceeded 25% within 3 h and was essentially complete within 7 days. CONCLUSION: In the University Hospital of Kuopio, newborns have been treated with nifedipine suspension prepared from tablets, and preliminary experiences with administration of suspension have been encouraging. It may also be possible to apply this methodology to other medicines used in paediatrics.