Fluorometric determination of magnesium in serum with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone

This simple, rapid, sensitive fluorometric method for determining magnesium is based on formation of a fluorescent complex of magnesium with 2-hydroxy-1-naphthaldehyde salicyloyl-hydrazone in alkaline ethanolic medium (lambda ex = 420 nm, lambda em = 460 nm). The detection limit of the method is 0.05 microgram/L (2 nmol/L) and the relative standard deviations (CVs) are 0.8%, 1.1%, and 2.5% at magnesium concentrations of 50, 5.0, and 0.50 microgram/L, respectively. The standard curve is linear from 0 to 250 micrograms/L (0-10 mumol/L). We investigated the chemistry of the reaction, and have applied the method to determine magnesium in 100-fold-diluted, otherwise untreated serum samples. Within-run CVs for the method were 1.7%, 1.1%, and 1.3% at mean magnesium concentrations of 15.7, 26.4, and 36.2 mg/L, respectively. Day-to-day CVs were 2.7%, and 3.6% at mean magnesium concentrations of 10.1 and 25.5 mg/L, respectively. Samples from 96 hospitalized patients in intensive-care units were analyzed by the proposed method (y) and by an automated colorimetric method involving Magon sulfonate reagent (x). Linear regression analysis of the results yielded: y = 1.01x - 0.04 (r = 0.982, Sxy = 0.90).     

尿素酰基裂解酶酶偶联法测定血清总钙

目的 介绍尿素酰基裂解酶酶偶联测定血清总钙的方法。方法 双试剂两点法；缓冲液为三乙醇胺（TEA，pH8.0,200mmol/L）；试剂I：含NaHCO326.0mmol/L,NADPH 0.4mmol/L,α－酮戊二酸8.0mmol/L，尿素酰基裂解酶115U／L，GLDH43．0kU/L；试剂Ⅱ：含NaCO3 26.0mmol/L,ATP6.2mmol/L。结果 方法线性范围为0．5－5．0mmol/L，批内和批间平均变异系数分别为2．13％和2．69％。平均回收率为102．7％ 。本法（Y）与邻甲酚酞络合酮法（OCPC法，X1）比较：r＝0．981，Y1＝1．02X1－0．021，与偶氮胂Ⅲ法（X2）比较：r＝0．978，Y＝0．99X2＋0．03。血清胆红素高达300μmol/L，血红蛋白4g/L，镁2.5mmol/L，NH4^ 2.0mmol/L,Trig 8.0mmol/L对本法无明显干扰。结论 本法具有简便、准确、快速，适用于自动分析等特点。     

Modified magnesium method in the aca III: elimination of interference by bilirubin

Bilirubin interferes with the Du Pont magnesium method in the aca at 510 nm. We determined that bilirubin concentrations in serum samples up to 380 mg/L did not affect the absorbance measured in the aca III at 540 nm, and therefore we modified the Du Pont setting of spectrophotometer for the magnesium method to 540 and 600 nm. Accuracy and precision of the modified method were comparable with the unmodified method and with atomic absorption spectrophotometry. For comparison, we analyzed serum samples with normal (n = 37) and increased (n = 22) bilirubin concentration with the modified method in the aca III (y) and in the atomic absorption spectrophotometer (x). The results (range 0.56-1.25 mmol/L) were in good agreement (x = y = 0.84 mmol/L, D(x - y) = -0.001, SD = 0.004, t = 0.238, P greater than 0.50) and bilirubin did not interfere with the modified method.     

Simple and sensitive determination of urea in serum and urine.

In this method for serum and urinary urea determination, the same reagent is used without predilution of urine samples. The method is based on the pH increase resulting from the ammonia released by urease hydrolysis of urea. o-Cresolphthalein complexone is used to monitor the pH change colorimetrically. Urea concentration and absorbance at 570 nm are linearly related for concentrations as great as 600 mmol/L for urine samples and 100 mmol/L for serum. There are no clinically significant interferences from physiological substances or drugs, and precision and accuracy are excellent (CV approximately 2%, except at very low concentrations in serum; analytical recovery was 99% in urine, 100% in serum). Results by this method (y) and by the Astra method (x) for urine correlated well (y = 0.991x - 2.87, Sy/x = 9.21, r = 0.994), as did the results by this method and by the total enzymatic method (x') for serum (y = 1.002x' + 0.192, Sy/x' = 0.598, r = 0.997). This method is applicable to automated as well as manual instruments, and one-reagent or two-reagent formats can be used.     

Magnesium content of mononuclear blood cells

The magnesium content of mononuclear blood cells may provide a better assessment of intracellular magnesium or total body magnesium status than does measurement of magnesium in plasma or erythrocytes. We describe a method for determining this, and report results for 20 normal volunteers. The mononuclear cells are separated with a discontinuous Ficoll-Hypaque gradient, washed, centrifuged at 400 X g, and lysed by sonication in 10 mmol/L NaCl. The magnesium in the cell lysate, with 2.93 g of added lanthanum oxide per liter, is determined by atomic absorption spectrometry. The mean mononuclear cell magnesium content in our volunteers was 70.7 (SD 14.1) fg per cell and 9.29 (SD 1.85) mg/g of DNA. The CVs for the determinations of magnesium, DNA, and cell count were 3.0%, 5.0%, and 8.7%, respectively. There was a significant correlation (r = 0.67, p less than 0.001) between results by the two methods of expressing magnesium content of mononuclear cells. However, by either method there were no significant correlations among results for magnesium concentration of mononuclear cells, plasma, or erythrocytes.     

脲酶结合邻甲酚酞络合剂显色检测血清及尿液尿素含量

目的　建立一种实用的血清和尿液中尿素含量的比色测定方法。方法　脲酶水解尿素产生氨(NH3),使溶液pH升高,以邻甲酚酞络合剂(OCPC)作为pH指示剂,波长570nm吸光度变化与尿素浓度成正相关。结果　该法线性范围血清1.00～200mmol/L,尿液10.0～1000mmol/L;检测界限血清0.36mmol/L,尿液2.19mmol/L。精密度分析血清批内CV1.13%～2.78%,批间CV1.81%～3.91%,总CV2.13%～4.80%。尿液批内CV0.68%～1.72%,批间CV0.96%～2.24%,总CV1.17%～2.82%。平均回收率血清100%,尿液102%。该法(Y)与酶速率法(X)比较相关性良好(血清Y＝1.048X+0.511,r＝0.998,S     

Revised calibration of the Reflotron cholesterol assay evaluated

We evaluated the Boehringer Mannheim (B.M.) Reflotron Total Cholesterol "dry-chemistry" method after its recalibration in 1987. Reports in the literature up to 1986-1987 of a negative bias (up to -10%) in the method prompted a revision of the factory-set calibration of the Reflotron. For this, B.M. prepared a new set of calibrators with 12 different concentrations of cholesterol. We checked in two ways whether accuracy had been achieved: (a) The values assigned to the calibrators by B.M. were checked with the manual Abell-Kendall Reference Method (MAK) performed in an official Reference Center. These were shown to be correct. (b) Concurrently, a direct comparison was made by analyzing 200 fresh samples of human serum. Reflotron cholesterol values obtained for these samples proved to be accurate, meeting the current World Health Organization/Centers for Disease Control criterion of maximum bias less than or equal to 5%. Orthogonal regression analysis yielded the following correlation: Reflotron = 0.985 MAK + 0.238 mmol/L (y = ax + b). Reflotron mean = 6.26 mmol/L; MAK mean = 6.09 mmol/L. SDa = 0.015 mmol/L; SDb = 0.120 mmol/L, and r = 0.989.     

Enzymic assay for oxalate in unprocessed urine, as adapted for a centrifugal analyzer

In this automated modification of the oxalate decarboxylase method, oxalate can be measured (12 per hour) in acidified but otherwise unprocessed urine. Standard curves are linear up to at least 2.5 mmol/L. When 0.50 mmol of oxalate was added per liter to samples of 18 patients' urines, a mean analytical recovery of 98.5% (SD 3.6%) was obtained. Within-series CVs were 3.4 and 1.0%, between-series CVs 7.3 and 2.7% (n = 15) for oxalate concentrations of 0.31 and 0.61 mmol/L. The lower limit of detection is 25 mumol/L. Concentrations measured with this "direct" method correlated well (r = 0.95) with those measured after precipitation with calcium and ethanol and resolubilization in dilute sulfuric acid. For 17 healthy volunteers the mean urinary excretion of oxalate was 0.37 (SD 0.14) mmol/24 h.     

酶法测定血清钠离子的评价

目的评价酶法测定钠离子的性能。方法评价血清钠离子酶法试剂的准确性、精密度、开盖稳定性、线性、与电极法的相关性、回收率及抗干扰性。结果酶法测定钠离子的变异系数(CV)<2%,定标周期能够维持5 d,准确度满足朗道质控品要求,线性范围达到100～160 mmol/L,与电极法具有很好的相关性[相关系数(r)=0.989 9],回收率为100.9%,在胆红素≤600μmol/L、甘油三酯≤20 mmol/L、维生素C≤0.5 g/L、血红蛋白≤10 g/L、铵离子≤25 mmol/L、钾离子≤10 mmol/L、镁离子≤5 mmol/L、钙离子≤5 mmol/L、锌离子≤50 mmol/L、铜离子≤50 mmol/L对钠离子测定无明显干扰。结论钠离子酶法试剂有较好的重复性、准确性和稳定性,线性良好,抗干扰能力强,能满足临床使用要求。     

A sensitive immunochemiluminescence assay for human serum amyloid A protein

We describe an immunochemiluminescence assay for human plasma serum amyloid A protein (SAA) in which specific rabbit polyclonal antibodies against synthetic peptides are used. The detection of the antigen-antibody reaction at 425 nm is based on a brief emission of light by a luminophor component (signal) in response to chemical energy. The working range of the assay covers plasma SAA concentrations from 5 to 100 micrograms/L. The lower detection limit is 5 micrograms/L, the within- and between-assay CVs are less than 12%. Bilirubin, cholesterol and triglyceride in final concentrations of up to 220 mumol/L, 8.1 mmol/L and 2.68 mmol/L, respectively, do not interfere with the assay. Results were correlated with those obtained by the enzyme-linked immunosorbent assay using the same antibodies (r = 0.95; p less than 0.001; n = 50). This method is inexpensive, simple and easily automated.     

The theophylline method of the Abbott "Vision" analyzer evaluated

We evaluated the analytical performance of the Abbott "Vision" analyzer for theophylline measurement. The within-day precision (CV) was 1.8% and 3.1% at theophylline concentrations of 15.2 and 25.2 mg/L, respectively; between-day precision was 3.5% and 4.8% at 14.9 and 24.4 mg/L, respectively. Bilirubin (143 mg/L) and triglyceride (7.4 g/L) did not interfere, but hemoglobin caused lower values for apparent theophylline, the magnitude of the decrease being proportional to the hemoglobin concentration. At the cutoff concentration of 1 g/L programmed into the instrument by the manufacturer, hemoglobin reduced the theophylline value by less than 10%. Results by the Vision method (y) compared well with those by the "TDX" procedure (x): r = 0.98, y = 0.978x - 0.270 mg/L. The Vision method gave comparable theophylline values for serum, plasma, and whole-blood samples. We also validated analytically and clinically that capillary blood samples collected by finger stick can be used interchangeably with blood samples collected by venipuncture for monitoring theophylline therapy.     

New enzymatic assay for serum urea nitrogen using urea amidolyase

We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on hydrolysis of urea by the enzyme. In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4). Then in the presence of urea amido-lyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum. The concentra-tion of ammonium ion formed was determined by adding GLDH to produce NADP(+) in the presence of 2-oxoglutarate and NADPH. We then monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion on this assay was eliminated by adding glyco-letherdiamine-N, N, N', N'-tetraacetic acid to the reaction system. The with-in-assay coefficient of variations (CVs) of the present method were 1.80-3.76% (n = 10) at 2.8-19.0 mmol/L, respectively. The day-to-day CVs were 2.23-4.59%. Analytical recovery was 92-115%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammo-nium ion, or calcium ion did not affect this assay system. The correlation be-tween values obtained with the present method (y) and those by another enzy-matic method (x) was 0.997 (y = 1.02x - 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of -0.18 +/- 0.86 mmol/L [(values by reference method - that of present method) +/- SD] using the Bland-Altman technique. J. Clin. Lab. Anal. 17:52-56, 2003.     

毛细管电泳紫外法检测重度创伤患者血清谷氨酰胺含量的实验研究

目的 探讨重度创伤患者血清中谷氨酰胺(glutamine,Gln)含量准确、简便、快速的检测方法.方法 选择我院重度创伤10例(创伤组),分别在术前1d及术后5、10d采集静脉血制备血清.选择同期健康体检10例为对照组,同法制备血清.以四硼酸钠(20 mmol/L,pH 9.33)、十二烷基硫酸钠(166 mmol/L)和乙腈按10:10:3配置缓冲液,异硫氰酸苯酯为衍生剂,在电压15 kV、紫外吸收波长254 nm下用直径75 μm、长58 cm的右英毛细管检测两组血清Gln含量.结果 Gln浓度在0.03 ～2.00 mmol/L范围内线性关系良好,相关系数为0.9901;精密度为3.14％,平均回收率为92.99％～ 110.28％;最低检测限浓度为0.01 mmol/L.对照组Gln含量0.79 mmol/L,创伤组术后5d时Gln出现相对最低点,且术前1d及术后5、10 d血清Gln浓度均低于对照组,差异有统计学意义(P＜0.05).结论 该毛细管电泳紫外法快速、准确而简便,灵敏度高,重复性好,能有效分离检测重度创伤患者血清中Gln含量.     

Kinetic measurement of the combined concentrations of acetoacetate and beta-hydroxybutyrate in serum

This is an automated method for the kinetic measurement of the combined concentrations of acetoacetate and beta-hydroxybutyrate in a single channel of the "Multistat III" centrifugal analyzer. Acetoacetate is first reduced with high concentrations of NADH by catalysis with 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). This reaction mixture is diluted with excess NAD+. The endogenous beta-hydroxybutyrate and that resulting from acetoacetate are then measured kinetically. Comparing the combined concentration of acetoacetate and beta-hydroxybutyrate (y) with the sum of acetoacetate and beta-hydroxybutyrate measured as described by Hansen and Freier (Clin Chem 1978;24:475) (x) yielded the relationship: y = 0.99x - 0.57 (r = 0.93, n = 25). The run-to-run CVs for low (5 mmol/L) and high (15 mmol/L) acetoacetate controls were 12% and 6%, respectively. The method is useful for determining the concentration of ketone bodies in 2-microL samples of serum of patients with diabetic ketoacidosis. The sensitivity can be increased to determine ketone body concentration in nonketotic individuals by increasing sample volume to 10 microL.     

Comparison of ultracentrifugation and a precipitation method for high-density lipoprotein cholesterol quantitation in insulin-dependent diabetic patients

We compared sodium phosphotungstic acid and magnesium chloride precipitation method for high-density lipoprotein (HDL) cholesterol quantitation with the ultracentrifugation method in 64 insulin-dependent diabetic patients with plasma triglyceride less than 3 mmol/l. The cholesterol content of HDL after precipitation of very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) was 86% +/- 3% of the cholesterol content of HDL (q greater than 1.063) determined after ultracentrifugation at q = 1.063 (1.33 +/- 0.05 mmol/l vs 1.55 +/- 0.06 mmol/l; p less than 0.001). HDL cholesterol determined after precipitation closely correlated to HDL cholesterol determined after ultracentrifugation (r = 0.97; p less than 0.001). The absolute difference between the HDL cholesterol values obtained by the two methods was correlated to HDL cholesterol (ultracentrifugation) (r = 0.75; p less than 0.001), but it was not correlated to VLDL cholesterol, LDL cholesterol, triglyceride, HbA1c, blood glucose or serum albumin. LDL cholesterol calculated by use of Friedewald's formula was 108% +/- 4% of the cholesterol content of LDL (q = 1.019 to 1.063), determined after ultracentrifugation, but the calculated and the ultracentrifugally determined LDL cholesterol values were closely correlated (r = 0.98; p less than 0.001). These results suggest that during sodium phosphotungstic acid and magnesium chloride precipitation of plasma from diabetic patients, a constant fraction of HDL cholesterol is co-precipitated, resulting in a systematic difference in HDL cholesterol quantitation when compared with the ultracentrifugation method.     

Lithium determined in serum with an ion-selective electrode

We evaluated the performance of the lithium ion-selective electrode (ISE) in the Du Pont Na/K/Li analyzer. Lithium concentrations in 106 serum samples from patients being treated with lithium were measured in duplicate with the ISE and by flame photometry. The slope of the regression line for the two methods was 1.004 with a standard error of the estimate of 0.049 mmol/L (x = flame photometry, y = ISE). Lithium measurements by the ISE method in serum or aqueous standards were linear to greater than 2.0 mmol/L. Within-run CVs for low (0.31 mmol/L) and high (1.15 mmol/L) lithium controls were 5.9% and 1.7%, respectively (n = 20). Day-to-day CVs for the same controls were 9.8% and 3.3%, respectively (n = 20). There was no significant interference when the concentrations of sodium, potassium, calcium, or magnesium were varied, nor did intervening urinary lithium analyses affect the measurement of serum lithium. Results for lithium measurement in four serum-based survey materials compared well with results by isotope dilution/mass spectrometry.     

31P nuclear magnetic resonance and zero-point titration compared for measuring free magnesium concentration in erythrocytes.

Intracellular ionized magnesium concentrations ([Mg2+]i) were measured in erythrocytes by 31P nuclear magnetic resonance (NMR) and zero-point titration in 14 controls and seven patients with renal magnesium loss. The mean intracellular ionized magnesium concentration in controls measured by 31P NMR was 0.20 (SD 0.03) mmol/L cell water, compared with 0.55 (SD 0.12) mmol/L cell water by zero-point titration. Total erythrocyte magnesium content measured with the lysate method was 0.63 mmol/L cell water higher than estimated by 31P NMR, probably because not all magnesium complexes are fully visible to the NMR technique. We found a positive correlation between plasma ultrafiltrable magnesium and [Mg2+]i irrespective of the [Mg2+]i assay used. [Mg2+]i measured with 31P NMR correlated modestly but significantly with [Mg2+]i determined by zero-point titration (r = 0.58, P less than 0.02). Washing erythrocytes before the zero-point titration decreased the ATP content and the cell water fraction, which led to overestimation of [Mg2+]i by zero-point titration. Although absolute values for [Mg2+]i differ with the assay used, both methods determined significantly lower values for [Mg2+]i in patients with isolated renal magnesium loss.     

高效毛细管电泳技术同时检测随机尿中香草扁桃酸、高香草酸和肌酐含量

目的 建立高效毛细管电泳技术快速检测随机尿中香草扁桃酸(vanillylmandelic acid,VMA)、高香草酸(homovanillic acid,HVA)和肌酐(creatinine,Cr)的方法.方法 在毛细管区带电泳模式下,以120 mmol/L NaH_2PO_4-Na_2HPO_4(pH值6.80)为缓冲液,在非涂层石英毛细管(47 cm×75μm内径)中进行电泳.样品离心后直接稀释压力进样4 s,20 kV电压分离后通过二极管阵列检测器(DAD)检测(λ=200 nm).对该法进行系统的方法学评价后,测定健康成人及儿童随机尿标本各100份,建立成人与儿童尿VMA/Cr、HVA/Cr参考值.结果尿中VMA、HVA和Cr在13 min内出峰.VMA、HVA和Cr分别在0～500、0～500、0～4 000 μmol/L范围内呈良好的线性,相关系数(r)在0.997 2～0.999 1之间(P<0.01),检出限(S/N=3)分别为1.0、1.0和50.0 μmol/L.尿中VMA、HVA和Cr迁移时间的平均批内(n=10)变异系数(CV)分别为0.58%、0.56%和0.25%,平均批间(n=10)CV分别为0.95%、1.00%和0.48%;峰面积的平均批内(n=10)CV分别为3.78%、3.97%和2.76%,平均批间(n=10)CV分别为4.60%、4.08%和4.42%.VMA、HVA和Cr平均回收率分别为98.36%、93.56%和98.85%.儿茶酚胺、5-羟色胺和清蛋白等对测定结果无干扰.该法与HPLC法有较好的相关性,VMA、HVA浓度的相关系数(r)分别为0.954 9(P<0.01)和0.945 1(P<0.01).健康成人与儿童随机尿标本的VMA/Cr、HVA/Cr比值均呈偏态分布(n=100),以百分位数法建立其95%参考值,成人VMA/Cr、HVA/Cr分别为0～4.26和0～1.69(μmol/mmol),儿童VMA/Cr、HVA/Cr分别为0～10.39和0～4.31(μmol/mmol).结论 该法可同时检测随机尿中VMA、HVA和Cr,样品用量少,且稀释后直接进样,操作简便、快速,准确度和精密度高,易于自动化,是尿液VMA、HVA常规检测及相关疾病筛查的理想方法.     

Sensitive colorimetric assay for angiotensin converting enzyme in serum

A sensitive colorimetric procedure has been developed for the assay of angiotensin converting enzyme (EC 3.4.15.1) in serum. Serum (10 microL) is incubated for 30 min with hippuryl-glycyl-glycine as described earlier (Clin Chem 28: 1352-1355, 1982). After a Folin-Wu deproteinization, the liberated glycyl-glycine is derivatized with a borate-buffered (pH 9.3) trinitrobenzenesulfonate solution (60 mmol/L) to form trinitrophenyl-glycylglycine, the absorbance of which is read at 420 nm vs a serum blank. The linear range extends to an activity of more than 900 U/L of serum and the detection limit is less than 4 U/L. The mean activity for serum from 50 blood bank donors and 25 patients with active sarcoidosis was 281 (SD 77) and 693 (SD 81) U/L, respectively. The method demonstrates good precision (CVs less than 2.8%) and correlates well (r = .99) with results from a "high-pressure" liquid chromatographic procedure for determining hippuric acid. In addition, the proposed method is widely applicable, involving only commonly available apparatus.     

Use of a competitive inhibitor in a kinetic enzymatic method for measuring ethanol in serum

We describe a kinetic enzymatic method for ethanol in serum, based on the use of pyrazole, a competitive inhibitor for alcohol dehydrogenase (EC 1.1.1.1). The method is rapid and is easily automated for the Cobas Bio centrifugal analyzer. No specimen pretreatment is necessary and the total reaction time is 120 s. The standard curve is linear up to 140 mmol/L. The within-run CV was between 1.9% and 3.5%; the between-run CV ranged from 2.6% to 4.1%. Mean analytical recovery of ethanol added to serum was 100.2%. We compared results of the kinetic enzymatic method with those from a gas-liquid chromatographic (GLC) method and another commercial alcohol dehydrogenase method (TDx REA Ethanol; Abbott Diagnostics). Linear regression analysis gave the following equations: kinetic = 0.991GLC - 0.354 mmol/L (r = 0.992, n = 110) and kinetic = 0.998TDx - 1.741 mmol/L (r = 0.993, n = 70). No interference from methanol or isopropanol was seen.