A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method

BACKGROUND: Several methods for measuring concentrations of lysophosphatidic acid (LPA), a lipid mediator, have been reported to date. However, these methods are not routinely used because most of them require specialized instrument and a complicated protocol. METHODS: We developed a novel LPA assay using enzymatic cycling. LPA in a sample is hydrolyzed with lysophospholipase to glycerol-3-phosphate, followed by enzymatic cycling using glycerol-3-phosphate oxidase and glycerol-3-phosphate dehydrogenase. Amplified concentrations of hydrogen peroxides, a product of the enzymatic cycling, are then colorimetrically measured. RESULTS: This method was specific for LPA, being insensitive to the presence of phosphatidic acid or lysophosphatidylcholine. The within-run and between-run CVs were 1.31-1.32% and 0.73-1.03%, respectively. The recoveries of exogenous LPA added to plasma were 100.3-101.6%. In males, LPA concentrations (mean+/-S.D.) of human serum and EDTA-plasma were 0.41+/-0.14 and 0.08+/-0.02 micromol/l, respectively. In females, they were 0.41+/-0.12 and 0.09+/-0.02 micromol/l, respectively. CONCLUSIONS: This novel colorimetric assay for determination of LPA using enzymatic cycling is simple and highly sensitive. It can be used with an automatic analyzer. It may also be useful for further studies of the biological functions of LPA as well as clinical applications in various disorders.     

Multicentre evaluation of an enzymatic method for creatinine determination using a sensitive colour reagent

A new enzymatic colorimetric method for the determination of creatinine in serum and urine (Creatinine PAP, Cat. No. 839434 and 836885, Boehringer Mannheim GmbH, Mannheim, FRG) was evaluated in 16 clinical chemistry laboratories and the manufacturer's testing laboratory. The test is based on the enzymatic degradation of creatinine and its reaction products by creatininase, creatinase and sarcosine oxidase. The H2O2 produced by the oxidation of sarcosine is determined using a modified Trinder reaction. The test can be carried out either manually or in mechanized analysers which enable the pipetting of a starter reagent to be made. The following results were obtained: Depending on the analyte concentration (range 40 to 1240 mumol/l), medians for the coefficients of variation were: 4.6-0.9% within-run and 6.4-2.8% between-day. At 546 nm the linear measuring range extended from 13 mumol/l (detection limit) to 1780 mumol/l, at 510 nm from 9 to 890 mumol/l. Recoveries in aqueous and human serum based standards as well as method comparisons with Fuller's earth methods and an enzymatic UV test indicate a high accuracy of this new enzymatic method in serum and urine. No interference was observed with haemolysed and lipaemic sera. This also applied to anticoagulants and to 36 drugs at therapeutic concentrations, with the exception of calcium dobesilate, which led to decreased values. Icteric samples containing 120-310 mumol/l bilirubin invariably led to decreased creatinine values (10-50 mumol/l lower). In a collaborative study substantially better interlaboratory agreement was observed with the new colorimetric enzymatic test than with the comparison methods (enzymatic UV test and various Jaffe procedures). In conclusion, this new enzymatic colorimetric test permits a precise and specific determination of creatinine in serum and urine. It makes a considerable contribution to improving the interlaboratory comparability of creatinine determinations and is suitable for routine use.     

肌酐酶法测定的两点法

建立了实用的肌酐酶法测定方法，通过实验将比色测定终点法改为两点法，使其适合自动分析，并节省工具酶。本法的线性为０－２７４９μｍｍｏｌ／Ｌ，精密度：手工操作批内ＣＶ〈３％，总ＣＶ〈５％。三种分析仪的批内ＣＶ在０．３４％－１．４４％间，回收率９９．６６％－１００．９％。     

F-DAOS色原的酶法测定血清肌酐试剂盒的评价

目的根据美国临床实验室标准化委员会(NCCLS)相关文件,评价N乙基N(2羟3磺丙)3,5二甲氧4氟苯胺(F DAOS)酶法肌酐(Cr)试剂盒。方法采用F DAOS酶法试剂盒测定血清Cr,并对精密度、线性范围、干扰因素及相关性进行分析。结果精密度批内变异系数(CV)为1.24%～2.15%,批间CV为2.33%～3.95%,总CV为2.52%～4.27%,线性达2870μmol/L,r=0.996。与Roche同类试剂对照,r=0.9894。上海市临床检验中心质控血清测定偏差值≤2.06%。胆红素<684μmol/L、血红蛋白<5.0g/L、维生素C<500mg/L时对本法无干扰。参考值:男性为55.8～136.2μmol/L,女性为36.8～111.2μmol/L。结论采用F DAOS色原的酶法测定血清Cr的方法特异性好,精密度高,结果准确,有一定推广价值。     

F-DAOS色原的酶法测定血清肌酐试剂盒的评价

目的根据美国临床实验室标准化委员会(NCCLS)相关文件,评价N-乙基-N-(2-羟-3-磺丙)-3,5-二甲氧-4-氟苯胺(F-DAOS)酶法肌酐(Cr)试剂盒.方法采用F-DAOS酶法试剂盒测定血清Cr,并对精密度、线性范围、干扰因素及相关性进行分析.结果精密度批内变异系数(CV)为1.24%～2.15%,批间CV为2.33%～3.95%,总CV为2.52%～4.27%,线性达2 870 μmol/L,r=0.996.与Roche同类试剂对照,r=0.989 4.上海市临床检验中心质控血清测定偏差值≤2.06%.胆红素＜684 μmol/L、血红蛋白＜5.0g/L、维生素C＜500 mg/L时对本法无干扰.参考值男性为55.8～136.2μmol/L,女性为36.8～111.2 μmol/L.结论采用F-DAOS色原的酶法测定血清Cr的方法特异性好,精密度高,结果准确,有一定推广价值.     

The effects of peritoneal dialysis solutions on peritoneal host defense.

Conventional peritoneal dialysis fluid (PDF) is a bioincompatible solution owing to the acidic pH, the high glucose concentrations and the associated hyperosmolarity, the high lactate concentrations, and the presence of glucose degradation products (GDPs). This unphysiologic composition adversely affects peritoneal host defense and may thus contribute to the development of PD-related peritonitis. The viability of polymorphonuclear leukocytes, monocytes, peritoneal macrophages, and mesothelial cells is severely depressed in the presence of conventional PDF. In addition, the production of inflammatory cytokines and chemoattractants by these cells is markedly affected by conventional PDF. Further, conventional PDF hampers the recruitment of circulating leukocytes in response to an infectious stimulus. Finally, phagocytosis, respiratory burst, and bacterial killing are markedly lower when polymorphonuclear leukocytes, monocytes, and peritoneal macrophages are exposed to conventional PDF. Although there are a few discrepant results, all major PDF components have been implicated as causative factors. Generally, novel PDF with alternative osmotic agents or with alternative buffers, neutral pH, and low GDP content have much milder inhibitory effects on peritoneal host defense. Clinical studies, however, still need to demonstrate their superiority with respect to the incidence of PD-related peritonitis.     

Value of an enzymatic assay for the determination of serum ceruloplasmin

The serum concentration of the copper protein ceruloplasmin has been an important diagnostic indicator of Wilson's disease (WD). It is widely quoted that 95% of people with WD have low serum ceruloplasmin concentrations. Current evidence suggests that a normal serum ceruloplasmin concentration is more common in patients with WD, particularly those with liver disease, perhaps in part because of the routine use of an immunologic assay. This assay might indicate a normal level of ceruloplasmin when the enzymatic activity is lower. Enzymatic activity is the biologically relevant parameter. We compared the immunologic measurement with the enzymatic assessment of oxidase activity in patients with liver or neurologic symptoms of unknown origin in whom WD was considered in the differential diagnosis. Although a strong correlation of ceruloplasmin protein concentration with oxidase activity was observed in controls, this was not the case for these patients. Twelve patients, presenting with various types of hepatic disease, demonstrated a weak correlation between ceruloplasmin protein concentration and oxidase activity. Ten percent of patients with neurologic symptoms ( n = 41) had low ceruloplasmin concentrations and oxidase activity, and another 8% had normal ceruloplasmin concentrations associated with low oxidase activity. Although the enzymatic method is preferred for its biologic relevance, ceruloplasmin analysis is not a reliable diagnostic parameter for the diagnosis of WD in patients with liver disease. An important use of the ceruloplasmin oxidase assay is in the follow-up of patients with WD. Ceruloplasmin oxidase activity was undetectable in sera from patients with WD who were undergoing long-term chelation therapy, suggesting an early sign of copper depletion and a need for subsequent monitoring for symptoms of copper deficiency.     

血清游离脂肪酸酶法测定的方法学评价及生物学变异研究

目的评价血清游离脂肪酸(FFA)酶学比色法(简称酶法)的精密度和正确度,并且研究血清FFA个体间和个体内生物学变异。方法采用美国临床和实验室标准化协会(CLSI)EP5-A2和EP15-A文件评价酶法测定血清FFA的精密度和正确度。生物学变异的研究对象为13名健康志愿者,在6周时间内每2周测定1次血清FFA浓度,利用SAS9.1软件中MIXED分析过程计算个体间生物学变异和个体内生物学变异。结果精密度实验样品的血清FFA浓度总均数为0.796 mmol/L,批内、批间、日间和室内不精密度分别为2.15%、3.44%、3.67%和5.46%。测定3个室间质评样品的百分比偏差分别为24.20%、-0.90%、-1.50%。血清FFA个体间和个体内生物学变异分别为8.43%、19.36%。结论酶法测定血清FFA的精密度和正确度能满足相关实验室质量控制要求。血清FFA的个体间和个体内生物学变异可为制定实验室质量规范和临床课题的科研设计提供参考依据。     

The choice of dialysis solutions in pediatric chronic peritoneal dialysis: guidelines by an ad hoc European committee.

OBJECTIVE: To provide guidelines on choosing dialysis solutions for children on chronic peritoneal dialysis (PD). SETTING: European Paediatric Peritoneal Dialysis Working Group. DATA SOURCE: Literature on the application of PD solutions in children (Evidence), and discussions within the group (Opinion). CONCLUSIONS: Glucose is the standard osmotic agent for PD in children (Evidence). The lowest glucose concentration needed should be used (Opinion). Low calcium solution (1.25 mmol/L) should be applied, wherever possible, with careful monitoring of parathyroid hormone levels (Opinion).The use of amino acid-containing dialysis fluids can be considered in malnourished children, although aggressive enteral nutrition is preferred (Opinion). There is insufficient evidence documenting the efficacy of intraperitoneally administered amino acids (Evidence). When ultrafiltration and/or solute removal are insufficient, polyglucose solutions are a welcome addition to the treatment of children on nocturnal intermittent PD (Evidence). However, in the absence of any reported long-term experience with children, their use must be closely monitored (Opinion). Bicarbonate would appear to be the preferred buffer for PD in children, but more in vivo studies are required before it replaces the present lactate-containing solutions (Evidence/Opinion).     

A population-specific formula predicting creatinine excretion in continuous peritoneal dialysis.

OBJECTIVE: The Cockroft-Gault formula was shown to systematically overestimate the decline in creatinine excretion with age in continuous peritoneal dialysis (CPD) patients and is, therefore, not suitable for studying creatinine excretion.The purpose of the present study was to develop and test a population-specific formula predicting average creatinine excretion in CPD. METHODS: Creatinine excretion in urine plus dialysate was measured in 925 CPD patients. Forty patients were excluded because of evidence of noncompliance. The remaining 885 subjects were randomly grouped into a derivation group (n = 432) and a validation group (n = 453). Stepwise multiple linear regression models were used to predict creatinine excretion in the derivation group. The candidate variables, chosen because they were previously shown to be predictors of creatinine excretion in CPD, included weight (W), age (A), gender (G), diabetes (D), and interaction terms between these four variables. Estimates of creatinine excretion from the best-fit regression formula (CrExcr1) and from the Cockroft-Gault formula (CrExcr2) were compared to creatinine excretion (CrExcr) in the validation group. RESULTS: The best-fit regression model in the derivation group included all four candidate variables (W, A, G, D), but no interaction terms. This model was as follows: CrExcr1 = 302.150 - 4.380A + 171.234G - 39.041D + 11.730W (r2 = 0.477, p < 0.001). In the validation set, CrExcr = -15.795 + 0.988CrExcr1 (r2 = 0.447, p < 0.001), and CrExcr = -303.823 + 0.732CrExcr2 (r2 = 0.340, p < 0.001). When the differences between measured and predicted creatinine excretion did not take into account the sign of each individual difference, CrExcr - CrExcr1 = 201 +/- 156 mg/24 hours, and CrExcr - CrExcr2 = 235 +/- 174 mg/24 hr (p < 0.001) in the validation group. When the sign of the difference was taken into account, CrExcr - CrExcr1 = -28 +/- 149 mg/24 hr, and CrExcr - CrExcr2 = 63 +/- 295 mg/24 hr (p < 0.001). CONCLUSIONS: A population-specific formula predicting creatinine excretion in CPD was derived.This formula has greater accuracy than the Cockroft-Gault formula and can be used in studies of creatinine excretion in CPD.     

Comparison between the enzymatic vitros assay for creatinine determination and three other methods adapted on the Olympus analyzer

To evaluate the relationship between the enzymatic Vitros assay for creatinine determination and other methods, we determined creatinine concentration in 400 heparin samples. Plasma creatinine level was successively determined on the Vitros 750 analyzer (Johnson & Johnson Co., Rochester, NY) and on the Olympus AU2700 analyzer (Olympus France, Rungis, France), using three reagents in the same assay: Olympus-Jaffé and two enzymatic commercial kits-Crea Plus (Roche Diagnostics, Meylan, France) and Enzymatic Creatinine (Randox, Mauguio, France). Comparison of Jaffé and enzymatic measurements of plasma creatinine levels revealed a high correlation when considering all values ranged from 20-1000 micromol/l (r > 0.99). However, for values <60 micromol/l, enzymatic reagents provided best results. Enzymatic methodology is a better clinical choice for the accurate measurement of creatinine, especially for neonates, pediatrics, and hematology units. Because analytical performance and the costs of Randox creatinine were satisfactory for our laboratory, this method, adapted on the Olympus analyzer, was chosen for routine determination of creatinine levels. According to the Valtec protocol (8), no interferences such as hemolysis, lipemia, or bilirubin were detected for Enzymatic Creatinine Randox.     

Fluid and solute transport using different sodium concentrations in peritoneal dialysis solutions.

BACKGROUND: Fluid and sodium balance is important for the success of long-term peritoneal dialysis. Convective transport is the major determinant for sodium removal during peritoneal dialysis using conventional dialysis solutions. However, recent studies showed that lower sodium concentration in dialysate could significantly increase sodium removal by increasing the diffusion gradient, thereby increasing diffusive transport. In the present study, we investigated the influence of the sodium concentration gradient on the diffusive transport coefficient, K(BD) for sodium. METHODS: A 4-hour dwell study was done in Sprague-Dawley rats using 25 mL 5% glucose (NS), 5% glucose + 0.3% NaCl (LS), 5% glucose + 0.6% NaCl (MS), or 5% glucose + 0.9% NaCl (HS), with frequent dialysate and blood sampling. Radiolabeled human albumin (RISA) was added to the solution as an intraperitoneal volume marker. The peritoneal fluid and sodium transport characteristics were evaluated. RESULTS: Significant ultrafiltration (both net ultrafiltration and transcapillary ultrafiltration) was observed in each group despite the osmolality of the 5% glucose solution being slightly lower than the plasma osmolality. There was no difference in peritoneal fluid absorption rate and direct lymphatic absorption among the four groups. With the sieving coefficient for sodium set to 0.55, a significantly higher K(BD) for sodium was found in the NS compared to the HS group. The K(BD) for sodium was 0.21+/-0.01, 0.20+/-0.01, 0.17+/-0.01, and 0.09+/-0.01 mL/min for the NS, LS, MS, and HS groups, respectively. The K(BD) values for glucose were significantly lower in the NS and LS groups compared to the MS and HS groups. CONCLUSIONS: Our results suggest that (1) sodium concentration may affect peritoneal sodium K(BD)--as the sodium concentration gradient increased, the K(BD) decreased; (2) 5% glucose solution could induce significant peritoneal ultrafiltration in normal rats despite its initial hypo-osmotic nature, this was due to the significantly lower glucose transport rate than sodium transport rate; and (3) a lower dialysate sodium concentration may decrease peritoneal glucose absorption.     

Advanced glycosylation end-products in diabetic rats on peritoneal dialysis using various solutions.

OBJECTIVE: To evaluate and compare the effects of glucose-based solutions to those of icodextrin with respect to peritoneal transport characteristics and advanced glycosylation end-product (AGE) formation in the peritoneal membrane in a diabetic rat model of peritoneal dialysis (PD). DESIGN: Thirty-three male Sprague-Dawley rats weighing between 275-300 g were divided into five groups: group C (n = 6), control rats implanted with a catheter but not dialyzed; group D (n = 5), diabetic rats implanted with a catheter but not dialyzed; group G (n = 7), diabetic rats implanted with a catheter and dialyzed with standard 2.5% glucose solution for daytime exchanges and 4.25% glucose solution for overnight exchanges; group H (n = 8), diabetic rats implanted with a catheter and dialyzed with standard 2.5% glucose solution for daytime exchanges and 7.5% icodextrin solution for overnight exchanges; group I (n = 7), diabetic rats implanted with a catheter and dialyzed with 7.5% icodextrin solution for all exchanges. Dialysis exchanges (25 mL per exchange) were performed three times daily for a period of 12 weeks. Tissue sections were stained using a monoclonal anti-AGE antibody. One-hour peritoneal equilibration tests (PET) were performed every 4 weeks for comparison of transport characteristics. RESULTS: The level of immunostaining was lowest in group C and highest in group G. Significant differences in immunostaining were seen between group C and group G (p < 0.001), group C and group H (p = 0.001), and group C and group I (p < 0.05). Significant differences were also found between group G and group D (p < 0.05), and between group G and group I (p < 0.05). Over time, the ratio of glucose concentration after 1 hour to glucose concentration at instillation (D/D0) decreased and the dialysate-to-plasma ratio (D/P) of urea increased. Significant differences in D/D0 glucose and D/P urea were found between group C and group H (D/D0: 0.40 +/- 0.01 vs 0.35 +/- 0.01, p < 0.05; D/P urea: 0.87 +/- 0.03 vs 0.97 +/- 0.02, p < 0.05). CONCLUSIONS: These results suggest that AGE formation is lower with the use of peritoneal dialysis solution containing icodextrin than with glucose-based solution. We conclude that use of icodextrin may help to slow the deterioration of the peritoneal membrane, prolonging its use for dialysis.     

Peritoneal transport in peritoneal dialysis patients using glucose-based and amino acid-based solutions.

OBJECTIVE: To evaluate peritoneal transport of fluid and solutes in continuous ambulatory peritoneal dialysis (CAPD) patients using amino acid (AA)-based versus glucose-based dialysis solutions. METHODS: Using iodine-labeled human serum albumin ((125)I-HSA) as intraperitoneal volume marker, peritoneal transport was investigated in a group of 20 clinically stable patients (11 females and 9 men, age 53 +/- 15 years) on CAPD for 15 - 101 months. Two paired 4-hour dwells, one with 1.36% glucose and one with 1.1% AA dialysis solution, were performed in each patient. Intraperitoneal dialysate volume, fluid absorption rate, and diffusive mass transport coefficients (K(BD)) and sieving coefficients (S) for glucose, creatinine, urea, potassium, and total protein were estimated for each dwell study. Dwell studies with AA solution were used to estimate K(BD) values for individual AAs. RESULTS: Intraperitoneal dialysate volume was higher for AA solution in comparison with glucose solution due to the higher osmolality of the AA solution. No statistically significant difference was found for K(BD) or S for creatinine, urea, potassium, or total protein in the dwell studies with either solution, whereas K(BD) for glucose was higher with AA than with glucose solution. Mean values of K(BD) of AA were similar but with high standard deviation, reflecting inter-individual variations in peritoneal transport rate. CONCLUSION: Our results indicate that the AA peritoneal transport rate is strongly dependent on transport characteristics of the individual peritoneal membrane.