Isoelectric focusing of human tissue alkaline phosphatase isoenzymes in agarose gel

Human tissue alkaline phosphatases obtained by butanol treatment of tissue homogenates were subjected to isoelectric focusing in agarose gel. Inactivation of alkaline phosphatases by ampholytes was prevented by incorporating zinc into the gel prior to electrofocusing and to the authors' knowledge this has not been reported before. The addition of zinc did not alter the pH gradient and the method was reproducible. Ampholytes from two different manufacturers differed in their requirements for zinc in preventing inhibition of alkaline phosphatase. The method is more rapid than previously described methods and is very sensitive.     

Simultaneous analysis of microheterogeneity of immunoglobulins and serum protein fraction using high-voltage isoelectric focusing on six cellulose acetate membranes

A systematic detection method for the single performance of cellulose acetate (CA) membrane isoelectric focusing to detect six different types of information on protein abnormalities was developed. High-voltage isoelectric focusing was carried out on six layers of CA membrane using a thermoelectric cooling apparatus. After electrophoresis, the proteins on the top, the third, the fourth, the fifth, and the bottom CA membrane were transferred to a polyvinylidene difluoride (PVDF) membrane by a simple contact printing procedure to detect IgM, κ-chain, λ-chain, IgA, and IgG, respectively. Each PVDF membrane revealed the microheterogeneity of these immunoglobulins using specified anti-serum and enzyme immunostaining. The second CA membrane was stained with Coomassie brilliant blue G250 to detect serum protein patterns. All stained membranes showed clear electrophoretic patterns of immunoglobulin microheterogeneity. By our method, immunoglobulin abnormalities in serum could be screened out using six different types of information obtained simultaneously. J. Clin. Lab. Anal. 11:220–224, 1997. © 1997 Wiley-Liss, Inc.     

粒细胞集落刺激因子诱导血液病人粒细胞中碱性磷酸酶同工酶基因表达的研究

目的：探讨粒细胞集落刺激因子（G－CSF）和视黄醇对正常组，真性红细胞增多症（PV），慢性粒细胞白血病（CML）中碱性磷酸酶同工酶（ALP）基因表达的诱导作用，并寻找何种同工酶的基因表达占主导地位。方法：应用半定量逆转录－聚合酶链反应（RT－PCR），并以β-actin作内对照，检测了20例正常对照者，11例PV初治患者和23例CML初治患者ALP基因的表达，以及G－CSF和视黄醇对其表达的诱导。结果：诱导前，骨型ALP（BALP）基因表达在正常对照组和PV组占主导地位，PV组的ALP基因表达显著高于对照组，未检测到CML组ALP基因表达；G－CSF诱导后，CML组出现ALP基因表达，仍以BALP为主，显著高于正常对照组，用G－CSF和视黄醇共同诱导后，CML组BALP表达明显增强，显著高于单独用G－CSF诱导；正常对照组和PV组在诱导前均无变化。结论：人类粒细胞主要以BALP基因表达为主；G－CSF和视黄醇对CML粒细胞中的ALP基因表达具有诱导作用。     

外周血中性粒细胞膜碱性磷酸酶诊断再生障碍性贫血的价值

目的探讨用流式细胞术检测再生障碍性贫血(aplastic anemia,AA)患者外周血中性粒细胞膜碱性磷酸酶(m NAP)变化的临床意义。方法病例对照研究。收集2014年8月至2015年12月连云港市第一人民医院收治的初诊全血细胞减少患者198例,其中明确诊断为AA者52例,对照组为非AA导致的全血细胞减少患者146例和体检健康者100例,用流式细胞术检测m NAP,ROC曲线评价其对AA的诊断效能。结果 AA、非AA疾病对照组及健康人对照组m NAP检测结果[M(P25~P75)]分别为13 931(11 128,21 605)、1 875(1 119,3 119)、1 831(1 371,2 411)AB/c,AA组m NAP表达水平明显高于非AA疾病对照组及健康人对照组,差异有统计学意义(P0.01),而非AA组与健康人对照组比较差异无统计学意义(P0.05)。m NAP诊断AA的ROC曲线下面积为0.95,在最佳切点为6 388AB/c时,诊断AA的敏感性为90.90%(95%CI 0.83~0.96),特异性为96.10%(95%CI 0.86~0.99)。结论检测外周血m NAP值对AA的鉴别诊断可能具有重要的辅助价值。     

醋酸纤维薄膜法血清蛋白电泳的影响因素分析与对策

目的对醋酸纤维薄膜法(CA)血清蛋白电泳实验过程中各种影响因素进行分析,并采取相应措施,确保电泳结果达到预期效果。方法采用CA进行检测。结果对实验过程的影响因素进行分析并采取相应改进措施后,实验结果图谱清晰可见。结论通过对实验各种影响因素进行处理,可以使实验效果更佳。     

Urinary protein fraction in healthy subjects using cellulose acetate membrane electrophoresis followed by colloidal silver staining

We previously reported a rapid and highly sensitive colloidal silver staining solution suitable for the cellulose acetate membrane. This method was useful for detecting even very small amounts of urinary protein. In the present study, we examined urinary protein fractions in healthy subjects, using cellulose acetate membrane electrophoresis (CAE) with a highly sensitive colloidal silver staining, in an attempt to determine the clinical relevance of urinary protein fractions. Sixty unconcentrated spot urine specimens were analyzed by CAE and calculated by densitometry. All of the samples were separated into five fractions by CAE. The mean +/- 1 SD of the percentage of five fractions was 28.37 +/- 8.51 in albumin, 4.30 +/- 4.19 in alpha1-globulin, 14.41 +/- 6.14 in alpha2-globulin, 19.45 +/- 7.10 in beta-globulin, and 33.46 +/- 8.24 in gamma-globulin. The albumin/globulin (A/G) ratio was 0.41 +/- 0.17. These six items and the concentrations of total protein, albumin, and beta-N-acetyl-D-glucosaminidase (NAG) did not significantly differ between males and females. NAG is the marker of tubulointerstitial nephropathy. The results suggest that there are no gender-dependent differences in the urinary protein fractions of healthy subjects.     

Rapid and highly sensitive colloidal silver staining on cellulose acetate membrane for analysis of urinary proteins

In order to establish a rapid staining method sensitive enough to stain the protein bands on cellulose acetate membrane and suitable for the analysis of urinary proteins in healthy subjects, we conducted a detailed examination of Kovarik's method designed for the analysis of proteins in cerebrospinal fluid in detail. For purpose of our study, we changed the pH of the staining solution used in Kovarik's method to pH 4.4 by adding acetic acid. With this solution, our staining method revealed a degree of sensitivity about three times higher than that of the original method, but specific sensitivity for albumin and γ-globulin remained almost unchanged. The solution preparation we used was much more simple and the reagent much more stable than in the original method. Our method also showed better reproducibility and linearity than the original method. These results point to the suitability of this method for the analysis of low-concentration proteins, such as urinary proteins, in healthy subjects. © 1996 Wiley-Liss, Inc.     

Increased serum alkaline phosphatase activity: a possible indicator of renal damage.

Increased levels of serum alkaline phosphatase (ALP) (E.C.3.1.3.1.) were observed in 25 patients with various urological conditions involving the kidneys: malignancy, complicated nephrolithiasis, and surgical and percutaneous manipulations. Other possible sources for increased ALP level, mainly hepatic and osseous, were excluded by history, laboratory tests, and liver and bone imaging. Studies of isoenzymes of ALP did not show a distinctive pattern. ALP levels returned to the normal range by treating the underlying lesions involving the kidney: nephrectomy, complete removal of stones, or removal of nephrostomy. The increase in serum ALP activity may be derived from the injury to the brush border membrane of the renal tubular cells. Renal function impairment and contrast media induced nephrotoxicity may also be responsible for the increased serum ALP. Serum ALP may be a marker for involvement of the kidneys in pathological processes and an indicator of complete treatment. This clinical observation is worthy of further study.     

体外循环术后中性粒细胞膜碱性磷酸酶的变化

目的观察体外循环(CPB)后外周血中性粒细胞膜碱性磷酸酶(NAP)表达的变化规律以及与白细胞(WBC)计数、C反应蛋白(CRP)水平之间的关系。方法选择连云港市第一人民医院心脏外科CPB心脏手术患者20例为研究对象,分别于术前、术后即刻0 h、术后24 h、术后48 h、术后72 h、出院时留取血标本,应用血细胞分析仪进行白细胞计数、免疫比浊法检测CRP浓度、流式细胞术定量检测NAP表达。结果 NAP表达和CRP浓度在CPB术后0 h显著下降,随后显著升高,48 h达到高峰,72 h开始下降,出院时恢复术前水平。相关分析结果表明,NAP的表达与血清CRP呈正相关,r为0.957(P<0.001),高于与WBC计数相关性,r为0.608(P<0.01)。结论 CPB后NAP表达显著升高,其变化规律与血清CRP基本一致,可以作为CPB下全身炎症反应严重程度的一个预警指标,长时间的NAP升高可能预示着机体存在炎症。     

Analysis of an expanded width of albumin fraction by cellulose acetate membrane electrophoresis in IgA nephropathy urine before treatment

Cellulose acetate membrane electrophoresis with colloidal silver stain re-vealed that the width of the albumin fraction in IgA nephropathy (IgAN) urine before treatment was significantly expanded. This phenomenon was not shown in IgAN urine after treatment or in non-IgAN urine. There was a reverse correlation between the width of the albumin fraction and the albumin con-centration in IgAN urine. By immuno-fixation, Tamm-Horsfall protein (THP) was located in the same position as the albumin band in IgAN urine before treatment; however, in the urine of a healthy subject it was located in the same position as alpha(1)-globulin. By ELISA, the THP-albumin complex concentration in IgAN urine before treatment was significantly higher than in the other two diseases. The width of the albumin fraction and the sodium ion concentra-tion of the urine were significantly correlated. The THP/albumin ratio in IgAN urine before treatment was significantly higher than in the other two groups. This suggests that the characteristic expanded width of albumin found by immunofixation indicates a THP-albumin complex, and that the sodium concentration of urine is involved in the formation of this complex.     

Examination of the molecular diversity of alpha1 antitrypsin in urine: deficit of an alpha1 globulin fraction on cellulose acetate membrane electrophoresis

In the clinical field of nephrology, a noninvasive approach employing the analysis of electrophoretic patterns in urinary protein has been established. In this study a total of 52 urine samples with IgA nephropathy (IgAN), anti-neutrophil cytoplasmic antigen-associated crescentic glomerulonephritis (GN), and other types of GN were analyzed. Patients with high alpha1 globulin (alpha1G) fractions, which contained alpha1AT in cellulose acetate membrane electrophoresis (CAE), tended to have alpha1 antitrypsin (alpha1AT) of normal molecular weight (57 kDa and 49 kDa), while patients with a deficit alpha1G fraction tended to have alpha1AT of low molecular weight (<49 kDa) (P < 0.01). The alpha1G fraction was significantly higher in patients with IgAN, and there were significantly more patients with normal molecular weight alpha1AT compared to patients with other diseases (P < 0.01). The isoelectric point of alpha1AT with lower-weight molecules was more on the alkali side compared to higher-weight molecules in two-dimensional electrophoresis. Detecting changes in alpha1G fractions in CAE may support the differential diagnosis of IgAN from other types of GN.     

外周血中性粒细胞膜碱性磷酸酶在预测全身炎症反应综合征患者血液细菌感染中的价值

目的探讨中性粒细胞膜碱性磷酸酶(m NAP)在预测全身炎症反应综合征(SIRS)患者血液细菌感染中的价值。方法采用前瞻性研究方法,对进行血培养的SIRS患者149例(血培养阳性组59例,血培养阴性组90例),用流式细胞仪检测患者外周血m NAP,乳胶增强散射免疫比浊法和电化学发光法分别测定C反应蛋白(CRP)和降钙素原(PCT)水平,比较各组间差异及指标间的相关性。应用ROC曲线评价m NAP、PCT和CRP预测SIRS患者血液细菌感染的价值。结果血培养阳性SIRS组外周血m NAP、PCT和CRP水平中位数(四分位数)分别为13 683(9 825,18 605)AB/c、5.680(1.300,18.390)ng/m L和139.00(65.60,201.00)mg/L,均明显高于血培养阴性SIRS组7 297(4 600,10 363)AB/c、0.432(0.115,1.645)ng/m L、75.25(36.90,125.25)mg/L,差异均有统计学意义(P0.01)。m NAP、PCT和CRP预测SIRS血液细菌感染的ROC曲线下面积(AUC)分别为0.806、0.797和0.692,敏感性分别为66.1%、76.3%和61.0%,特异性分别为83.3%、74.4%和70.0%,阳性预测值(PPV)分别为72.2%、66.2%和58.1%,阴性预测值(NPV)分别为78.9%、79.8%和73.3%,m NAP和PCT联合检测时AUC为0.841(95%CI:0.774~0.908),敏感性和特异性分别为79.7%和78.9%。血培养阳性组m NAP水平与PCT呈显著正相关(r=0.509,P0.01)。结论 m NAP联合PCT检测对预测SIRS患者血液细菌感染具有较高的敏感性和特异性,有较好的临床应用前景。     

Comparison of polycarbonate and cellulose acetate membrane filters for isolation of Campylobacter concisus from stool samples

One thousand seven hundred ninety-one diarrheic stool samples were cultivated for Campylobacter spp. We found a high prevalence of Campylobacter concisus with use of a polycarbonate filter (n = 114) compared to a cellulose acetate filter (n = 79) (P < .0001). The polycarbonate filter is superior to the commonly used cellulose acetate filter for detection of C. concisus.     

Serum creatine kinase MM sub-band determination by isoelectric focusing: a potential method for the monitoring of myocardial infarction

Fresh myocardium homogenates analyzed by thin-layer isoelectric focusing revealed the presence of two prominent creatine kinase (CK; EC 2.7.3.2) sub-bands, MMO (pI 7.10) and MM1 (pI 6.88), in approximately equal proportion. While these forms represented together as much as 85% of the cellular MM fraction, they accounted only for viz. 2.2 and 27.7% of the total serum MM activity when measured 8 h before the CK peak in patients with myocardial infarction. Incubation of the isolated MMO and MM1 with normal human serum demonstrated that the former turned to MM1 within 5 h at 37°C; further changes affecting MM1 gave rise to other sub-bands, MM2 (pI 6.70), MM3 (pI 6.45), and MM4 (pI 6.25). In our patient population, these three forms represented more than 75% of the serum CK-MM activity at the CK peak; hence, soon after the enzyme release, the serum MM isoenzyme mainly consists of degradation products arising from the labile MMO and MMl. Among the two cellular forms, MMO was the best related to the total enzyme activities and the most efficient for differentiating the patients with left ventricular failure from the others during the entire survey period (F = 3.8, p < 0.05). Because its presence in the blood provides evidence for a very recent CK release from the tissues, serum CK-MMO determinations might be proposed for following the extension of the lesion after a myocardial infarct.