Simple enzymatic screening assay for ethylene glycol (ethane-1,2-diol) in serum

We present a novel method for the analysis of ethylene glycol (ethane-1,2-diol) in serum. This method employs the ability of glycerol dehydrogenase (EC 1.1.1.6) to oxidize ethylene glycol. Two measurements, five and 30 min after addition of enzyme, are used to circumvent interferences from endogenous glycerol and from propylene glycol (propane-1,2-diol). Ethanol does not interfere in the assay. The method requires commonly available equipment only, making it suitable for all emergency laboratories.     

Effects of dextran on five biuret-based procedures for total protein in serum

We evaluated the effect of dextran on values for total protein in serum as measured by the biuret method with five widely used automated instruments: the American Monitor Parallel; the Du Pont aca II; the Roche Cobas-Bio; the Kodak Ektachem 400; and the Beckman Astra 8. Dextran concentrations as great as 25 or 30 g/L had relatively little or no influence on total protein measurements by the latter three instruments. Dextran concentrations exceeding 6 g/L caused falsely low results with the aca, whereas the Parallel gave falsely high results when the dextran concentration exceeded 2 g/L. The aca total protein procedure could be protected from the interference by dextran concentrations up to 30 g/L by injecting 0.4-0.8 mL of ethylene glycol directly into the reagent pack before sampling. However, we could not eliminate the interference with the Parallel procedure by any simple means; we thus recommend that it not be used for measuring total protein in serum samples from patients who are being treated with dextran.     

Raspberry-like Pd3Pb alloy nanoparticles: superior electrocatalytic activity for ethylene glycol and glycerol oxidation

Pd₃Pb catalysts are one of the state-of-the-art catalysts for the electrooxidation of alcohols. Herein, raspberry-like Pd₃Pb catalysts are synthesized via a simple method. The materials are characterized using various physical techniques. The electrocatalytic behaviors of the products towards the oxidation of ethylene glycol and glycerol are investigated. Electrochemical results show that the raspberry-like Pd₃Pb nanostructure produces excellent electrocatalytic activity and stability towards the electrooxidation of ethylene glycol and glycerol in alkaline media, which endows the prepared nanostructure with promising potential in applications like fuel cells.

Raspberry-like Pd₃Pb nanoparticles are prepared and employed as electrocatalyst towards ethylene glycol and glycerol oxidation.     

不同添加剂对自制同型半胱氨酸质控品质量的影响

目的探讨不同添加剂对自制同型半胱氨酸(Hcy)室内质控品质量的影响。方法以混合人血清为基质,分别加入叠氮钠、乙二醇、丙三醇作为添加剂制备Hcy质控品,评价不同质控品的不精密度、开瓶稳定性和储存稳定性。结果 3种质控品的不精密度和开瓶稳定性均符合要求,以添加乙二醇的质控品不精密度和开瓶稳定性最佳;在储存稳定性方面,添加叠氮钠的质控品稳定期只有4个月,添加乙二醇、丙三醇的质控品稳定期均大于12个月。结论以乙二醇作为添加剂制备Hcy质控品,综合性能优于丙三醇和叠氮钠。     

Hydroxyurea interferes negatively with triglyceride measurement by a glycerol oxidase method

Measured triglyceride concentrations were extremely low (less than 100 mg/L) in the serum of some patients who were receiving hydroxyurea for myeloproliferative diseases. The assay being used to quantify triglycerides was a "cascaded" enzymatic method involving (a) lipase, to generate glycerol from triglycerides; (b) glycerol oxidase, to convert glycerol to glyceraldehyde, with generation of hydrogen peroxide; and (c) peroxidase, which acts on the hydrogen peroxide with subsequent coupled generation of a red-violet quinone (reagent system used in the Technicon RA-1000). Hydroxyurea added to serum samples appeared to inhibit the action of glycerol oxidase, with a stoichiometric relation to the concentration of substrate (a decrease of roughly 2.4 mmol/L in measured triglyceride per 1 mmol of hydroxyurea per liter). A different enzymatic assay for triglycerides, which involves glycerol kinase (Beckman Instruments) did not show this effect of hydroxyurea.     

甘油内空白法与GPO-POD法检测血清三酰甘油

目的 探讨甘油内空白法与甘油磷酸氧化酶(GPO)-过氧化物酶(POD)法测定生化质控血清中三酰甘油(TG)的差异.方法 采用两种方法测定7个不同品牌共17个批号生化室内质控血清和卫生部2006年常规化学室间质评样本三酰甘油,比较两法测定结果的异同.结果 甘油内空白法与GPO-POD法测定不同品牌不同批次生化室内质控血清三酰甘油的结果无明显相关,Roche与Leadman的TG结果可相差1倍以上;而Aalto和Bio Rad的结果非常相近.不同批次的卫生部室间质评样本结果与此类似.结论 部分品牌的生化质控血清含大量的游离甘油,两法测定质控血清的结果可能出现明显差异.     

Enzymatic measurement of serum glycerol using a COBAS-BIO centrifugal analyzer

An enzymatic method for the measurement of glycerol using glycerol kinase coupled to pyruvate kinase and lactate dehydrogenase was adapted to a COBAS-BIO centrifugal analyzer. Routine standardization is not required under the optimized conditions since the calculation factor derived from the measurement of pure glycerol standard was found to be identical to the theoretical value. Excellent correlation between the method and the DuPont ACA triglyceride method was obtained. The coefficients of variation for within-run and day-to-day precision were less than 4%. Only 2 microL of serum is required, making this an excellent means for monitoring therapeutic glycerol administration in premature infants with hydrocephalus. The method has the sensitivity to measure glycerol concentrations as low as 0.1 mmol/L. This, together with the rapid analysis time (28 specimens in 10 min), makes it suitable for the correction of glycerol interference in the measurement of serum triglyceride.     

A kinetic colorimetric procedure for quantifying magnesium in serum

We have developed a kinetic colorimetric procedure for determination of magnesium in serum. The magnesium-dependent enzyme glycerol kinase is used to phosphorylate glycerol to glycerol 3-phosphate, the latter being oxidized to dihydroxyacetone phosphate and hydrogen peroxide by glycerophosphate oxidase. The generated hydrogen peroxide is then reduced by peroxidase with the simultaneous oxidative coupling of 4-aminoantipyrine and 2-hydroxy-3,5-dichlorobenzenesulfonate, producing a red reaction product with an absorption maximum at 510 nm. The rate of color production is proportional to the concentration of the Mg X ATP complex, which is, in turn, proportional to the magnesium concentration in serum. This method is rapid and precise, avoids the use of expensive instrumentation, is easily automated, and results compare well with those by the Du Pont aca and manual Magon sulfonate methods.     

A simple enzymatic fluorimetric method for the determination of triglycerides in 10 microliters of serum

An assay for the determination of triglyceride (acyl glycerol) in microliter volumes of serum or plasma is described. The method is based on enzymatic hydrolysis of triglyceride by lipase from Rhizopus arrhizus followed by enzymatic fluorimetric assay of the glycerol so released. Under the conditions of the assay the enzymatic hydrolysis is complete for concentrations up to 5 mmol/l in less than 15 min at room temperature. The determination of free glycerol and glycerol released is based on the formation of dihydroxyacetone in the presence of NAD and glycerol dehydrogenase. The NADH which is formed in stoichiometric quantities is estimated fluorimetrically. In the presence of the ketone-trapping agent hydrazine the reaction goes to completion above pH 9.0. By this method acyl glycerol can be measured routinely in a 10 microliters sample of serum or plasma. The test is extremely sensitive compared to previously described methods and exhibits acceptable precision and reproducibility.     

An enzymic determination for serum phospholipid

A new colorimetric determination for serum phospholipid is described. Firstly, serum phospholipid is incubated with phospholipase C from Bacillus cereus, and then the released diglyceride and triglyceride are hydrolyzed completely to fatty acid and glycerol by lipoprotein lipase from Pseudomonas fluorescens. Secondly, the glycerol produced is enzymatically determined by glycerol dehydrogenase in the presence of NAD⁺, using phenazine methosulfatenitro blue tetrazolium as color reagents. The absorbance at 570 nm is recorded. The amount of the glycerol from phospholipid is calculated by subtracting the amount of glycerol from triglyceride from the amount of total glycerol. The present method requires only 20 μl of serum and a 40 min incubation and is highly reproducible. The results obtained show good correlation with those obtained by a chemical method (correlation coefficient, 0.925) or the phospholipase D-choline oxidase method (correlation coefficient, 0.936). These results strongly suggest that the proposed method can be utilized as a routine clinical test.     

Interconversion and comparison of the results of three methods for cholinesterase in serum

We investigated three methods for determination of cholinesterase (ChE) in human blood sera, using reference sera, sera of known ChE content, and serum variously diluted with deactivated sera. The methods were the Du Pont aca analyzer, the Pfizer "ChE-tel" method, and the Michel method with and without direct reagent-blank correction. With respect to precision and reproducibility the methods ranked: aca greater than Michel method corrected directly for reagent pH changes (Michel) greater than ChE-tel method greater than Michel method corrected indirectly for reagent pH changes. The interrelationships derived, in terms of each method's individual units were: Michel = (0.054 +/- 0.001) aca - (0.043 +/- 0.086), r2 = 0.947 ChE-tel = (4.44 +/- 0.30) aca - (5.11 +/- 0.99), r2 = 0.959 ChE-tel = (80.2 +/- 5.8) Michel + (0.028 +/- 0.240), r2 = 0.947 The ChE-tel method was subject to variations due to the quality of different kits. The aca results were validated by a collaborative laboratory study. Sera of known activity can be used effectively for accurate calibration.     

Massive Ethylene Glycol Poisoning Triggers Osmotic Demyelination Syndrome

Background

Ethylene glycol is a toxic organic solvent implicated in thousands of accidental and intentional poisonings each year. Osmotic demyelination syndrome (ODS) is traditionally known as a complication of the rapid correction of hyponatremia.

Objective

Our aim was to describe how patients with ethylene glycol toxicity may be at risk for developing ODS in the absence of hyponatremia.

Case Report

A 64-year old female patient was comatose upon presentation and laboratory results revealed an anion gap of 39, a plasma sodium of 150 mEq/L, a plasma potassium of 3.5 mEq/L, an osmolal gap of 218, an arterial blood gas pH of 7.02, whole blood lactate of 32 mEq/L, no measurable blood ethanol, and a plasma ethylene glycol concentration of 1055.5 mg/dL. The patient was treated for ethylene glycol poisoning with fomepizole and hemodialysis. Despite having elevated serum sodium levels, the patient's hospital course was complicated by ODS.

Conclusions

Rapid changes in serum osmolality from ethylene glycol toxicity or its subsequent treatment can cause ODS independent of serum sodium levels.     

Salicylate determined with a microcentrifugal analyzer, and compared with Du Pont aca, trinder, and liquid-chromatographic methods

We describe a new automated method for measuring serum salicylate in the Multistat III microcentrifugal analyzer. Ferric nitrate reagent and serum blanking are used. We compare this new method, the automated Du Pont aca method, and the manual Trinder method with a "high-performance" liquid-chromatographic method. The unblanked Trinder method had the poorest correlation (r = 0.980, Sy X x = 19.1) with the chromatographic method. The serum-blanked aca and Multistat III methods showed better correlation (r = 0.995, Sy X x = 9.5 mg/L, and r = 0.991, Sy X x = 13.0 mg/L, respectively) with the chromatographic method. However, we conclude that all three colorimetric methods give clinically useful results and that the increased time, expense, and expertise required for chromatographic salicylate analysis are difficult to justify in a routine clinical laboratory.     

Clearance of Ethylene Glycol by Kidneys and Hemcdialysis

Abstract

A patient with acute ethylene glycol poisoning was treated with ethanol administration and hemodialysis, and his renal function remained consistently normal. Serial measurements of serum and urine levels of urea, creatinine, ethylene glycol, and ethanol were performed to compare the relative contributions of the hemodialyzer and the patient's kidneys in clearing ethylene glycol from the blood. Simultaneous measurements of the serum osmolal gap (corrected for ethanol) and anion gap were correlated with these data. Mean renal clearance of ethylene glycol was 27.5 ± 4.1 ml/min, with a fractional ethylene glycol excretion of 19.8 ± 1.5%. This was lower than the mean urea clearance of 89.4 ± 11.0 ml/min and fractional urea excretion of 66.0 ± 7.8%. Hemodialyzer clearance of ethylene glycol was 156 ml/min. There was a nearly exact correlation between the serum ethylene glycol level and the corrected osmolal gap (r = 0.998, p < 0.01). The calculated renal elimination half-life of ethylene glycol was 18 hr. We conclude that with a moderate diuresis, the normal human kidney contributes significantly to the removal of ethylene glycol from the blood. Corrected serum osmolal gap provides a nearly exact approximation of the serum ethylene glycol level and is a useful therapeutic guide.     

Inhibition of hepatic triglyceride formation by clofibrate

The effect of clofibrate (CPIB) on hepatic glycerolipid formation has been studied in vivo and in vitro in the rat. Feeding 0.25% CPIB in laboratory chow significantly reduced serum triglyceride levels by 6 hr and concomitantly decreased the rate of glycerol-(14)C incorporation into hepatic and serum glycerides, in vivo. These changes persisted for at least 14 days. A similar decrease in serum triglyceride and glycerol incorporation into hepatic glycerides was observed in rats fed high glucose diets containing 0.25% CPIB. Serum glycerol was reduced by feeding CPIB for 14 days. The formation of diglyceride and triglyceride from (14)C-sn-glycerol-3-P by rat liver homogenates was inhibited by addition of 1-40 mM CPIB to the reaction mixture. These results suggest that CPIB reduces hepatic glycerolipid synthesis, possibly by inhibition of one or more reactions in the esterification of sn-glycerol-3-P. This change may account for the early fall in serum triglyceride. At later time periods, serum glycerol levels fall and in some experiments, hepatic triglyceride content increases. Therefore, it is likely that additional metabolic alterations may contribute to the sustained hypotriglyceridemic effects of CPIB.     

The Effect of High-Carbohydrate Diets on Liver Triglyceride Formation in the Rat

The effect of feeding diets containing 75% glucose or fructose on liver triglyceride formation in the rat was studied by both in vivo and in vitro techniques. The results were compared with those from control rats fed laboratory chow.Both high-sugar diets increased the capacity for triglyceride formation from sn-glycerol-3-P by rat liver homogenates and correspondingly increased incorporation of [1,3-(14)C]glycerol into hepatic triglyceride by the intact animal.These independent measures of hepatic triglyceride production changed with a similar time-course characteristic for each diet. The 75% fructose diet produced a greater increase in both determinations, reaching a maximum after 11 days.Despite the increase in hepatic triglyceride formation by both high-sugar diets, only the 75% fructose diet resulted in a consistent and sustained increase in serum triglyceride. This results most probably from differences in the fractional rate of serum triglyceride removal between the two groups.When serum triglyceride removal was inhibited by administration of Triton WR-1339, both high-sugar diets increased incorporation of [1,3-(14)C]glycerol in serum triglyceride in vivo and increased serum triglyceride level above that in control rats.     

Effect of serum lyophilization on the rate constants of enzymatic methods for measuring cholesterol

We determined the equilibrium absorbances and rate constants for two enzymatic methods, aca (DuPont) and RA-1000 (Technicon), used in determining cholesterol in reconstituted lyophilized serum. The lyophilized materials included two serum pools, three control materials, a College of American Pathologists' survey material, and Standard Reference Material no. 909. We calibrated the reagents with aca standards for cholesterol (DuPont). The difference in the mean concentrations of cholesterol (aca - RA-1000) was -0.09 g/L overall and was not statistically significant by analysis of variance. The mean rate constant for all materials was 0.23 min-1 for the aca and 1.42 min-1 for the RA-1000, significantly different (P less than 0.001). Lyophilization causes lower results for the aca method than for the Ra-1000, because the reaction rate for the aca method is slower and has not reached equilibrium when the final absorbance reading is made.     

Ethylene glycol poisoning: quintessential clinical toxicology; analytical conundrum

Ethylene glycol poisoning is a medical emergency that presents challenges both for clinicians and clinical laboratories. Untreated, it may cause morbidly or death, but effective therapy is available, if administered timely. However, the diagnosis of ethylene glycol poisoning is not always straightforward. Thus, measurement of serum ethylene glycol, and ideally glycolic acid, its major toxic metabolite in serum, is definitive. Yet measurement of these structurally rather simple compounds is but simple. This review encompasses an assessment of analytical methods for the analytes relevant for the diagnosis and prognosis of ethylene glycol poisoning and of the role of the ethylene glycol metabolites, glycolic and oxalic acids, in its toxicity.     

Albumin activation of urinary amylase as determined with the Du Pont aca.

Protein activation of urinary alpha-amylase (EC 3.2.1.1) activity was observed during an evaluation of the Du Pont aca procedure for the determination of urinary alpha-amylase. This activation effect became constant for urinary albumin concentrations exceeding 1.50 g/liter. It is recommended that urinary alpha-amylase be analyzed with sufficient albumin added to maximize this effect. The aca alpha-amylase procedure is compared to an amyloclastic method for both serum and urine analysis. Expected ranges are presented for the aca method for serum and urinary amylase, amylase clearance, and the amylase clearance/creatinine clearance ratio.     

Paraquat Poisoning–Prolonged Excretion

Abstract

Monitoring of individuals poisoned with ethylene glycol involves analysis of ethylene glycol in serum. The objective of this procedure was to validate a colorimetric and gas chromatographic procedure for glycolic acid in serum. The colorimetric procedure requires no sophisticated instrumentation and has been shown to be specific for glycolic acid. A gas chromatographic procedure has also been developed involving methyl derivatization of glycolic acid and the internal standard (propionic acid). These methods have been used for the analysis of serum specimens from ethylene glycol poisoned patients. Glycolic acid has been recognized as the major toxic agent in ethylene glycol poisoning but current methods available do not allow analysis in a clinically relevant turnaround time. These two procedures allow glycolic acid quantitation by procedures readily set up in most clinical toxicology laboratories.