Effects of temperature on measurement of alkaline phosphatase activity

We examined the effects of temperature on the activity and steady-state kinetic properties of alkaline phosphatase (EC 3.1.3.1). Purified isoenzymes from human liver, intestine, and placenta were used, as was human serum, and the enzyme from porcine kidney. Phosphatase activity was estimated by two different assay techniques. For all isoenzymes, apparent Michaelis constants for the substrate 4-nitrophenyl phosphate decreased with increased temperature; Km at 37 degrees C was typically half that determined at 25 degrees C. All enzymes of human origin exhibited similar linear Arrhenius relationships over the range examined, 20-37 degrees C (Ea of 30-36 kJ X mol-1). The porcine kidney enzyme obeyed an Arrhenius relationship that was slightly, but significantly, different from the isoenzymes of human origin. Temperature relationships based upon Arrhenius behavior and individual activity measurements are presented. For human alkaline phosphatases, they differed by no more than 10%.     

Placental alkaline phosphatase isoenzyme in quality control materials may be a source of variability in alkaline phosphatase activity

ObjectivesTo determine the cause of discrepancies in the alkaline phosphatase (ALP) activities of quality control (QC) materials in two different analyzers using IFCC method.Design and methodsALP activities of patients' samples and QC materials (QC1 and QC2 from Bio-Rad) measured using TBA-200FR and Synchron LX-20 analyzers were compared and isoenzyme electrophoresis was done. Fractional mixing of bone or liver ALP with placental ALP was performed. ALP activities of QC materials were measured in TBA-200FR with pH-modified reagents.ResultsALP activities of QC materials were significantly lower in TBA-200FR than in LX-20. Placental ALP comprised 57% of QC1 and 95% of QC2. Higher placental ALP proportion in the mixture with bone or liver ALP resulted in lower ALP activities in TBA-200FR. The discrepancy in ALPs of QC materials decreased when measured with pH-modified reagents.ConclusionPlacental ALP in QC materials and differences in reagents' pH caused the discrepancy in ALP activities.     

骨型碱性磷酸酶在骨肿瘤诊断中的价值

目的检测恶性骨肿瘤、良性骨肿瘤及转移性骨肿瘤患者血清中的总碱性磷酸酶(TALP)和骨型碱性磷酸酶(BALP)活性,以观察TALP和BALP在骨肿瘤的鉴别诊断中的价值及在监测恶性骨肿瘤治疗中的作用。方法对2004年积水潭医院骨肿瘤科收治的40例恶性骨肿瘤、40例良性骨肿瘤及14例转移性骨肿瘤患者,采用法国临床化学会推荐的速率法测定血清TALP及酶联免疫吸附(ELISA)方法测定BALP水平。结果恶性骨肿瘤组的TALP活性为(325·55±356·36)U/L,BALP活性为(177·19±240·67)U/L,与同龄对照组TALP(120·70±70·59)U/L,BALP(61·30±44·45)U/L比较有统计学意义(P<0·05)。转移性骨肿瘤组的TALP(100·14±35·39)U/L,BALP(32·93±15·68)U/L,与同龄对照组TALP(57·64±11·06)U/L,BALP(19·92±7·53)U/L比较有显著性统计学意义(P<0·01)。而良性骨肿瘤组与同龄对照组相比无统计学意义(P>0·05)。同时恶性骨肿瘤与其他两组比较也都有统计学意义(P<0·01),并且恶性骨肿瘤组患者化疗前后的结果也有统计学意义(P<0·05)。结论血清TALP和BALP是反映骨肿瘤骨代谢的一个灵敏而简便的生化指标,并对骨肿瘤的鉴别诊断及疗效观察具有一定的临床意义。作为骨肿瘤的检测指标,血清BALP比TALP更为特异和敏感。     

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。     

Comorbid diseases in patients on dialysis: the impact on anemia.

Patients on dialysis frequently present with a multitude of comorbid diseases. Many of these conditions can either directly aggravate preexisting anemia, or lead to acute or chronic inflammatory or infectious conditions that can lower hemoglobin levels. Awareness of these conditions and their compounding effect on anemia can help nurses when interpreting the results of longitudinal trends in hemoglobin and enable them to intervene proactively to minimize the effect of these conditions on hematological parameters.     

Clinical evaluation of high-performance affinity chromatography for the separation of bone and liver alkaline phosphatase isoenzymes

The newly described high-performance (HPLC) affinity chromatography method for the separation of human bone and liver alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes was clinically evaluated. The improved resolution of bone from liver isoenzyme and lower detection limit was achieved by conjugation of wheat-germ lectin (WGL) to a diol-bonded silica gel column, stepwise elution with N-acetylglucosamine (NAG) and post column derivatization using para-nitrophenyl phosphate substrate. To establish a reference interval, we measured bone ALP in 86 healthy women, ages 33 to 95 years. The normal reference interval is described by a piecewise linear regression on age (R2 = 0.20, P less than 0.01). For women less than or equal to 45 years, bone ALP, U/l = 8.495. For normal women between ages of 45 to 55 years, bone ALP, U/l = -12.765 + 0.472* age. If age greater than or equal to 55 years, then bone ALP, U/l 13.219. In all 10 patients with primary biliary cirrhosis, serum bone ALP levels were elevated. In addition, sera from 43 patients with diverse metabolic bone diseases were evaluated. As expected, the sera from all 6 patients with Paget's disease and 2 with osteolytic metastasis had bone ALP activity which was greater than 3 standard deviations (SD) from the mean. In all 10 patients with hypoparathyroidism, bone ALP levels were depressed. Only 1 of the 9 patients with glucocorticoid excess and 2 of the 7 patients with primary hyperparathyroidism had elevated bone ALP when compared to the 95% confidence interval for the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intestinal alkaline phosphatase in serum of patients on maintenance haemodialysis

The sensitivity of serum alkaline phosphatase to inhibition by l-phenylalanine was used to investigate the percentage of intestinal alkaline phosphatase in normal subjects, hospital patients and in a group of patients on maintenance haemodialysis. The patients on haemodialysis showed an increased percentage of intestinal alkaline phosphatase relative to the other two groups. On several occasions a number of the haemodialysis patients showed elevated levels of total alkaline phosphatase which by inhibition and electrophoretic studies appeared to be predominantly of intestinal origin. Possible explanations of these findings are discussed.     

Simulated weightlessness and bone metabolism: impairment of insulin effect on alkaline phosphatase activity in bone tissue.

The effect of simulated weightlessness on bone alkaline phosphatase was investigated after skeletal unloading for up to 4 days. The skeletal unloading was designed by using the model of hindlimb hang in rats. The femoral-diaphyseal fragments obtained from rats bred with skeletal unloading were cultured for 24 h at 37 degrees C in 5% CO2/95% air in Dulbecco's Modified Eagle Medium (high glucose). The bone alkaline and acid phosphatase activity were significantly decreased by skeletal unloading. When the bone tissue was cultured with synthetic [Asu1,7] eel calcitonin (3 and 30 nM), the hormone caused a significant increase of alkaline phosphatase activity in the bone tissues from rats with normal and skeletal-unloading. In culture with insulin (1.0 and 10 nM), skeletal unloading impaired the effect on insulin to increase bone alkaline phosphatase activity. Meanwhile, the culture with zinc sulfate (10 and 100 microM), which can increase bone protein synthesis, caused a remarkable elevation of alkaline phosphatase activity in the bone tissues form rats with normal and skeletal-unloading. Insulin (10 nM) did not alter the zinc effect. These findings suggest that the skeletal unloading with hindlimb hang causes the impairment of insulin's effect to increase alkaline phosphatase activity in the femoral diaphysis of rats, although the effects of calcitonin and zinc were not altered.     

Clinical usefulness of alkaline phosphatase isoenzyme determinations.

1. We report on the clinical usefulness of alkaline phosphatase isoenzyme determinations using a combined chemical inhibition method on 731 patient serum specimens exhibiting elevated (greater than 350 U/L) alkaline phosphatase (AP) activity. 2. The relative percentages of the organ-specific alkaline phosphatase activities were computed on the basis of three independent assays: total activity, activity in the presence of 10 mMl-phenylalanine, and activity in the presence of 3.1 M urea. 3. Gamma-glutamyl transferase (GGT) activity assays were also performed on the same specimens. Using an upper reference limit of 30 U/L for GGT and comparing the GGT results with the percent liver AP, we found that the GGT results were 91% sensitive and 60% specific. 4. We conclude that AP isoenzyme determinations are very useful in identifying the organ source(s) responsible for elevated AP values. 5. The reference ranges for several age groups in relation to the adult population and their significance are presented.     

Bone alkaline phosphatase measured with a new immunoradiometric assay in patients with metabolic bone diseases

We have investigated the clinical utility of a new quantitative two-site radioimmunometric assay specific for bone alkaline phosphatase (B-ALP) in 219 healthy control subjects and in 264 patients with various metabolic bone diseases. B-ALP was compared with total alkaline phosphatase (T-ALP) and with osteocalcin (BGP). B-ALP increased linearly with age in both sexes. In postmenopausal normal women B-ALP increased by 82% compared with premenopausal normal women, whereas the differences between pre- and postmenopausal women for T-ALP and BGP were 18% and 30% respectively. As assessed by Z -score, the highest values of B-ALP were found in patients with Paget's disease of bone, bone metastases or hyperparathyroidism and in patients on maintenance haemodialysis. In osteoporotic patients, B-ALP, but not T-ALP, showed a slight but significant ( P  < 0.05) difference compared with normal women. On the basis of bone turnover, osteoporotic patients were divided into two groups: high turnover and low turnover; B-ALP, like BGP, was significantly ( P  < 0.01) higher in patients with high turnover. In conclusion, B-ALP, measured by this new method, can be considered a sensitive marker of bone turnover and could be especially useful in identifying women at risk of developing osteoporosis.     

Clinical significance of serum high-molecular-mass alkaline phosphatase,alkaline phosphatase–lipoprotein-X complex,and intestinal variant alkaline phosphatase

This paper is a study to identify the clinical significance of high-molecular-mass alkaline phosphatase (ALP:E:C.3.1.3.1.), ALP–lipoprotein–X complex (LP-X) and intestinal variant ALP. We used cellulose acetate and agarose gels and techniques including wheat germ lectin, cetavlon-diethyl ether, thermostatability, neuraminidase and L -phenylalanine to improve the electrophoretic separation of the alkaline phosphatase isoenzymes. Patients' serum samples were electrophoresed from a diverse group of individuals ill with cholestasis, neoplastic disease metastatic to the liver, hepatocellular carcinoma, cirrhosis, diabetes mellitus, and chronic renal disease. Agarose gels provided better separation of ALP isoenzymes than cellulose acetate gels. The results also indicated that high-molecular mass ALP is present in patient's serum in conditions associated with cholestasis especially caused by hepatic malignancy. High-molecular mass ALP was frequently found to co-exist with the liver isoenzyme and LP–XALP complex. The intestinal variant was identified in patients with malignancy, cirrhosis, chronic renal disease and diabetes mellitus. Intestinal ALP coexisted concomitantly with a variant intestinal ALP. Intestinal variant ALP is most likely composed of intestinal ALP attached to a cellular membrane-binding domain, or may be an artifact produced by neuraminidase incubation © 1994 Wiley-Liss, Inc.     

Different distributions of human bone alkaline phosphatase isoforms in serum and bone tissue extracts

BACKGROUND: In vitro, bone alkaline phosphatase (BALP) is released from the osteoblast membrane with its glycosylphosphatidylinositol (GPI) anchor still attached (i.e., in an anchor-intact form); however, in vivo, BALP circulates as a variable mixture of anchorless isoforms, which can be identified by high-performance liquid chromatography (HPLC). Previous studies have shown that the relative abundance of these BALP isoforms in serum may be clinically useful for the diagnosis and management of metabolic bone disease. METHODS: In the current studies, we describe a method for the determination of anchorless BALP isoforms in extracts of bone and we present novel data on the conversion of anchor-intact to anchorless BALP by incubation with endogenous circulating GPI-specific phospholipase D (GPI-PLD). RESULTS: A 72-h extraction with 0.1% Triton X-100 released approximately 90% of the BALP activity from powdered bone. An average of 19% of this activity was anchorless, but essentially all of the activity could be converted to the anchorless form by incubation with partially purified GPI-PLD from human serum. Using HPLC, we detected four BALP isoforms (B/I, B1x, B1, and B2) in these GPI-PLD-treated extracts of bone. An additional BALP fraction was also detected in the samples during the initial phase of GPI-PLD treatment. CONCLUSIONS: The abundance of the BALP isoforms differed between bone and serum, particularly for the B/I isoform, which comprised, on average, 18% of the BALP in GPI-PLD-treated extracts of healthy bone tissue, but only 6% of the total BALP activity in serum from healthy individuals. Based on our recent finding of differences in the number of sialic acid residues between the BALP isoforms, we hypothesize that this difference between BALP isoforms in serum and extracts of bone is due to the different patterns of glycosylation, which results in different biological half-lives in the circulation. A preliminary application of our method to the extraction of BALP isoforms from a small number of human bone samples suggests that the method should be useful for studies of human skeletal site-specific and metabolic bone disease-specific differences in the amounts and distributions of the BALP isoforms in bone.