Determination Of Ethanol In Serum Research Articles

P52: Forensic alcohol analysis: Analytical requirements and problematics of interpretation

Introduction In Algeria, The legal limit of BAC is 0,2 g/L. This low rate justifies the use of sensitive and specific techniques to provide accurately results. The objectives are: 1 Compare results of BAC measured by an official method, gas chromatography (HS-CPG/FID) and an automatic method using an enzymatic test Emit ® II Plus, designed for the determination of ethanol in serum or plasma, and applicated for analyzing whole blood pretreated. 2 Evoke the problematic of forensic interpretation of the alcohol concentrations perfomed for forensic porposes when the mesured rates are technically validated and correctly reported. Methods The procedure adopted to compare the two methods involves a duplicate analyzing of 11 whole blood samples loaded with pure ethanol, by enzymatic method after deproteinization and HS-CPG/FID without any pretreatment. The results are evaluated to estimate differences between the two methods. The postmortem samples of blood are analyzed by these methods in the context of toxicological expertise. Results The application of the test Emit ® II Plus to whole blood after deproteination made of alcohol measurements by enzymatic method as reliable as those made by HS- CPG /FID with a coefficient of correlation equal 0.999. It is found that for concentrations lower than 0,55 g/L, the results of both methods have a high level of similarity. From 0,55 g/L, the enzymatic method tends to generate overestimation with an average of 0,13 g/L. These overestimations are tolerable insofar as the subject remains under penalty of repression whatever the level of alcohol reached when the measured rate exceeds 0,2 g/L. In the situation of analysis for alcohol in postmortem specimens, preanalytical error risks have been detected and complicated the interpretation of the alcohol concentrations. Conclusion This enzymatic method is an interesting alternative to the instrumental technique (GC) when the equipment is correctly calibrated and controlled and the results are correctly reported. However, the exclusive and uncritical use for forensic purposes is not recommended. Forensic toxicologists are called on to interpret the alcohol concentrations found in the specimens taking into account the risk of preanalytical errors.

Evaluation of Potential Lactate/Lactate Dehydrogenase Interference with an Enzymatic Alcohol Analysis

The Connecticut Department of Public Safety laboratory recently addressed a legal challenge to a hospital alcohol dehydrogenase (ADH)-based serum ethanol determination based on the suggestion of interference by lactate dehydrogenase (LDH)-catalyzed oxidation of lactate. Both ADH- and LDH-oxidations require NAD(+) (present in excess in the assay). NADH produced by LDH-catalyzed lactate oxidation in the assay is interpreted as derived from ethanol. Hepatic trauma was suggested as the basis for elevated levels of lactate and LDH. Clinical laboratory results were evaluated, specifically serum hepatic enzymes, ions, and anion gap. Aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) were 229 and 144 U/L, respectively (approximately 8x and 4x reference range midpoint values). Na(+), K(+), Cl(-), and CO(2) levels were 143, 3.0, 112, and 20 meq/L, respectively, yielding an anion gap of 8 meq/L (ref. range 8-15). Serum lactate contributes to "unmeasured anions"; hence, the anion gap was inconsistent with a significant lactate elevation. Based on the slight elevation of ASAT and ALAT, LDH levels were estimated to be elevated to no more than 10-fold. Calculation of the amount of LDH and ADH present in the ethanol assay suggest an ADH/LDH ratio of 200:1. Hence, contribution by lactate oxidation to the serum ethanol concentration in this case would have been negligible.

Detection of Recent Ethanol Intake With New Markers: Comparison of Fatty Acid Ethyl Esters in Serum and of Ethyl Glucuronide and the Ratio of 5-Hydroxytryptophol to 5-Hydroxyindole Acetic Acid in Urine

At present, recent ethanol consumption can be routinely detected with certainty only by direct measurement of ethanol concentration in blood or urine. Because ethanol is rapidly eliminated from the circulation, however, the time span for this detection is in the range of hours. Several new markers have been proposed to extend the detection interval, but their characteristics have not yet justified their use in routine clinical practice. We therefore investigated three new markers and compared their kinetics and sensitivities: (1) fatty acid ethyl esters (FAEEs) in serum, (2) ethyl glucuronide (EtG) in urine, and (3) the ratio of 5-hydroxytryptophol to 5-hydroxyindole acetic acid (5-HTOL/5-HIAA) in urine. Seventeen healthy men participated in a drinking experiment. Blood and urine samples were collected twice daily on three consecutive days and once daily on days 4 and 5. Ethanol concentration was determined by gas chromatography, FAEE levels, by gas chromatography with mass spectrometry, EtG concentration, by liquid chromatography-tandem mass spectrometry, and 5-HTOL/5-HIAA ratio, by high-performance liquid chromatography. The peak serum ethanol concentrations of the subjects ranged from 5.4 to 44.7 mmol/liter (mean +/- SD, 30.1 +/- 9.1 mmol/liter). In the case of the serum ethanol determination, 100% sensitivity was reached only immediately after the end of the drinking experiment, and in the case of FAEE levels and 5-HTOL/5-HIAA ratio, it tested for 6.7 hr after the end of the ethanol intake. Thereafter, these latter parameters declined until 15.3 hr (FAEEs) and 29.4 hr (5-HTOL/5-HIAA), subsequently remaining in a stable range until 78.5 hr without further decrease. In contrast, EtG concentration showed 100% sensitivity until 39.3 hr and thereafter decreased, falling to below the limit of quantification of 0.1 mg/liter at 102.5 hr. After moderate drinking, EtG in the urine proved to be a superior marker of recent ethanol consumption in healthy subjects. This is because EtG is a direct ethanol metabolite, it occurs in the urine only when ethanol has been consumed, and its sensitivity remains at the level of 100% for 39.3 hr.

Derivation and validation of a formula to calculate the contribution of ethanol to the osmolal gap

Study Objectives: We sought to evaluate the relationship between osmolal gap and serum ethanol level and derive a formula that can be used clinically to calculate the expected osmolal gap in the presence of ethanol. Some investigators have noted that the residual osmolal gap appears to increase as the ethanol level increases, and thus it is important to determine the exact relationship between these 2 values. Methods: In part 1, a convenience sample of emergency department patients undergoing serum ethanol determination had sodium, urea, and glucose levels and osmolality determined on the same blood sample, and values were prospectively recorded. Predicted osmolality excluding ethanol was calculated with the following formula: 2 Na (mEq/L) + (Urea [mg/dL])/2.8 + (Glucose [mg/dL])/18. The osmolal gap was determined by subtracting the calculated serum osmolality excluding ethanol from the measured serum osmolality. Linear regression analysis was then used to derive a formula for the relationship between ethanol and the osmolal gap. This formula was then prospectively validated on a second convenience sample of patients. In part 2, we repeated this experiment in vitro by adding known amounts of ethanol to serum. Results: We derived the formula to calculate the contribution of ethanol to the osmolal gap by using 98 observations. The mean ethanol level was 197.8 mg/dL (SD 138.5), with a range of 0 to 538.2 mg/dL. The relationship between ethanol and osmolal gap was linear, with a Pearson coefficient of correlation of 0.99. Linear regression analysis generated a model with the following formula: Osmolal gap=(Ethanol [mg/dL])/3.7 – 0.35 or, in SI units: Osmolal gap (mOsm/kg)=1.25 (Ethanol [mmol/L]) – 0.35 The 95% confidence interval (CI) for the multiplicative factor was 1/3.58 to 1/3.80 (or, in SI units, 1.21 to 1.28). The 95% CI for the additive constant was –2.19 to 1.50. We prospectively validated our formula on 128 patients. The mean residual osmolal gap for this group of patients was 0.84 mOsm/L (SD 5.65; range, –18.40 to 17.85 mOsm/L). The results of the in vitro experiments were similar. Conclusion: Our data suggest that the best formula for the calculation of the contribution of ethanol to osmolality is as follows: Ethanol (mg/dL)/3.7 or, in SI units: 1.25 (Ethanol [mmol/L]) [Purssell RA, Pudek M, Brubacher J, Abu-Laban RB. Derivation and validation of a formula to calculate the contribution of ethanol to the osmolal gap. Ann Emerg Med. December 2001;38:653-659.]

A Response to “Serum-Ethanol Determination: Comparison of Lactate and Lactate Dehydrogenase Interference in Three Enzymatic Assays”: Reply

A Response to “Serum-Ethanol Determination: Comparison of Lactate and Lactate Dehydrogenase Interference in Three Enzymatic Assays”: Reply

Hydroxyethylmethacrylate column reactors with immobilized glucose oxidase or alcohol oxidase. Liquid chromatographic determination of ethanol in serum

Glucose oxidase and alcohol oxidase were immobilized in column reactors packed with hydroxyethylmethacrylate-based sorbents modified with epoxy or vinylsulphate groups. The properties of the were tested in the determination of glucose and ethanol. The best results were obtained with a sorbent containing a high concentration of epoxy groups (900–1100 μ mol g −1). The limits of detection were 1.0 × 10 −5 mol 1 −1 for glucose and 8.6 × 10 −5 mol 1 −1 for ethanol. The linear dynamic ranges amounted to about three concentration decades. The calibration graphs exhibited a good linearity and the precision of measurement was satisfactory. The method was applied to the determination of ethanol in serum after chromatographic separation on a C 18 column.

Flow injection analysis—use of immobilised enzymes for the determination of ethanol in serum

Flow injection analysis—use of immobilised enzymes for the determination of ethanol in serum

Vapor pressure and freezing point osmolality measurements applied to a volatile screen.

This is a report of a rapid and precise screening procedure, developed for the determination of ethanol in serum using osmolality measurements. The osmolality of the patient is determined by freezing point method (freezing point osmometry) and dew point (water vapor pressure osmometry) method. The difference between freezing point osmolality and vapor pressure osmolality (delta osm) is due to the presence of volatiles in the serum, because the volatiles are not measured by vapor pressure osmometry. The amount of ethanol (mg/dL) in serum is estimated by multiplying delta osm by a factor of 4.2. As a comparison method, ethanol also is measured by a spectrophotometric alcohol dehydrogenase method. In addition, a significant difference between an osmometric alcohol assayed value and enzymatic spectrophotometric measurement indicates the presence of volatiles, other than ethanol. In addition to ethanol there is a linear relationship between osmolality and isopropanol or methanol when added in vitro to serum.

Determination of ethanol in serum by an enzymatic pms-int colorimetric method

A simple, rapid and accurate colorimetric method for the determination of ethanol in serum was studied. Ethanol is converted to acetaldehyde by alcohol dehydrogenase, and the NADH formed transfers its hydrogen through the phenazine methosulphate- p-iodonitrotetrazolium violet (PMS-INT) system to produce the red formazan which is stable and absorbs at 505 nm. The method correlates very well with an enzymatic-UV method, r = 0.98 ( p < 0.001). The precision (95% limits) of the method for samples ranging from 11 to 145 mg/dl was ± 6.6%, and the recovery averaged 99%.

Automated enzymic determination of ethanol in blood, serum, and urine with a miniature centrifugal analyzer.

We describe an automated spectrophotometric reaction-rate method for determination of ethanol in serum and urine with a miniature centrifugal analyzer. The theanol is selectively oxidized in the presence of alcochol dehydrogenase and NAD+ to form NADH, which is measured by the rate of change of its absorbance. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer-generated working curve based on aqueous ethanol standards. Blood, serum, or urine specimens need not be deproteinized. The method permits duplicate analysis of at least 30 samples per hour. Coefficients of variation and relative errors are about 2-3% for ethanol concentrations of 0.3-3.0 mug per 2 mul of sample. Analytical recovery of ethanol added to serum is 92-103% (average, 98.5%). Comparisons with distillation-oxidation, gas-chromatographic, and conventional enzymic procedures give satisfactory agreement.