Ethanol

Ethanol in Blood

Undoubtedly, the drug most frequently studied for in vitro stability in biological specimens is ethanol. Studies have been performed using specimens from living individuals and from autopsy cases. Both increases and decreases in ethanol concentration during storage have been reported. Mechanisms of ethanol loss include evaporation, chemical oxidation to acetaldehyde, and microbial consumption.

The most common source of in vitro ethanol production is microbial conversion of glucose, fatty acids or amino acids to ethanol. The vast majority of studies performed on ethanol stability have been on blood specimens. Autopsy specimens have been used to monitor ethanol production at room temperature. For examples Plueckhahn and Ballard obtained blood specimens from 50 autopsies and stored the specimens with and without preservatives. In 34 cases there was no significant production of ethanol after 10 days; the remaining 16 specimens showed significant ethanol production.

The average increase in ethanol concentration after 2 to 3 days was 0.030 ± 0.028 g/dL, but the average increase after 6 to 10 days was 0.048 ± 0.036 g/dL. I he maximum increase over 10 days was 0.13 g/dL. The presence of 1% sodium fluoride prevented in vitro ethanol formation after 10 days. In contrasts 0.1% mercuric chloride failed to prevent ethanol formation, and 1% mercuric chloride in many specimens produced a solid that precluded ethanol analysis. A subsequent study reported 7 cases in which storage of the blood at room temperature produced increases greater than 0.05 g/dL.

A maximum blood concentration of 0.14 g/dL was measured after 7 days. After postmortem blood specimens were stored at room temperature for 15 days, Christopoulos et al. found an average increase of 0.05 g/dL (range 0.02-0.10 g/dL) in & specimens that initially tested negative for ethanol. However, there was no consistent pattern in 14 blood samples that originally contained ethanol, some specimens showed increases in concentration, others showed decreases in concentration and the remaining specimens were within 10% of the original ethanol concentration. When the 6 negative specimens were refrigerated, 5 remained negative after 15 days.

When refrigerated, the 14 positive specimens showed variability similar to that demonstrated at room temperature; however, the magnitude of the changes was less when the blood was refrigerated. The addition of 1% sodium fluoride to blood specimens stored at room temperature decreased the variability in ethanol concentration during storage to the extent that most of the samples were ± 0.025 g/dL of their original concentrations after 45 days. The combination of 1% fluoride and refrigeration produced changes within 0.01 g/dL of the original concentration after 45 days. Olsen and Hearn looked at the change in alcohol concentrations in 32 paired postmortem blood and vitreous humor specimens. The blood was collected and stored in 50-mL polypropylene tubes containing sodium fluoride and potassium oxalate.

The vitreous humor specimens were stored in 10-mL Vacutaine tubes containing 25 mg of potassium oxalate and 26 mg of sodium fluoride. The specimens were stored under refrigeration and reanalyzed 5 to 6 years later. Decreases in ethanol concentration in both blood and vitreous humor specimens were observed. The average loss of ethanol in the blood was 0.06 g/dL a 35% loss. The average loss of ethanol in the vitreous humor was 0.01 g/dL, a 6.1% loss. They concluded that vitreous humor may be a more reliable specimen for reanalysis of ethanol after prolonged refrigeration. In addition to postmortem blood ethanol analysis, testing of suspected intoxicated drivers is also commonly performed. In these cases, the integrity of the blood sample tested can be critical to successful prosecution; therefore, many studies have been published discussing the stability of ethanol in these samples.

Glendening and Warmth stored fluoridated blood specimens at room temperature for time periods ranging from 1 week to 3 years before reanalysis. Of the 90 samples reanalyzed, 76 showed a decrease in ethanol concentration, 11 showed an increase and 3 showed no change. Changes observed up to the 2-month interval were not statistically significant. The average decrease after 1 to 3 years was 0.04 g/dL. Similar studies performed on refrigerated blood indicated decreases of less than 0.003 g/dL after 2 months and an average decrease of 0.032 g/dL after 10 months. Bradford reanalyzed 4,000 blood specimens containing 0.01% mercuric chloride stored at room temperature from 1 to 6 months; in no instance was an increase in blood ethanol concentration observed. Brown et al. studied 6 parameters that could affect ethanol concentration: time of storage (4 weeks vs 8 weeks), fluoride (Presence or absence), alcohol concentration (0.11 g/dL vs 0.22 g/dL), temperature (4 °C vs 17 °C) and container Polypropylene vs glass).

The 3 important factors that determined ethanol loss were times temperature, and fluoride. Potential contribution of headspace volume was not discussed. There was an increased loss of ethanol in specimens stored at a higher temperature for a longer time period in the absence of fluoride. Winek and Paul stored 32 blood samples under refrigeration and 42 samples at room temperature, and periodically reanalyzed these over 14 days. No preservatives were present in any of the samples No changes in ethanol concentration were observed in either group. Long-term storage of fluoridated blood specimens at room temperature was studied by Chang et al. Blood specimens from different containers were reanalyzed after 3 years (N = 37) and 6.75 years (N = 57).

The loss of ethanol after 3 years ranged from 0.003 to 0.037 g/dL with an average of 0.019 g/dL; the loss after 6.75 years ranged from 0.003 to 0.072 g/dL, win an average of 0.033 g/dL. In 32 cases in which the original tube was reanalyzed, an average loss of 0.061 g/dL was observed, derived from a loss range of 0.034 to 0.093 g/dL. Dick and Stone demonstrated that ethanol loss in blood contaminated by Pseudomonas species is not prevented by 16 sodium fluoride at 4 °C, nor at ambient temperature, but that 2% sodium fluoride did prevent this loss. Amick and Habben demonstrated that 2.5 mg/mL sodium fluoride can prevent the production of ethanol in blood inoculated with Saccharomyces cerevisiae at 4 °C and at 25 °C.

Ethanol in Urine

The stability of ethanol in urine has also been examined by several investigators. Blackmore inoculated 30 samples with different microorganisms known to produce ethanol. In none of these samples was more than 0.002 g/dL ethanol detected despite incubation at 37 °C for 18 to 96 h. Neuteboom and Zweipfemung studied the stabilizer of 38 fluoridated urine specimens stored at 10°G with subsequent reanalyses after 1, 4, and 12 months. Thirty-two specimens exhibited decreases in ethanol concentration after 12 months, but the remaining 6 specimens were unchanged. All decreases were under 896 with an average decrease of 4.5%.

Christopoulos et al. refrigerated 20 urine specimens, 10 containing ethanol and 10 without ethanol. No significant changes were observed after 30 days, regardless of the presence or absence of phenylmercuric nitrate as a preservative. When these same specimens were stored at room temperature for 30 days, 2 of the negative specimens had some ethanol production while the positive specimens without a preservative showed a reduction in concentration. The preservative prevented these losses at room temperature. Ball and Lichtenwalner reported on one urine specimen in which ethanol concentration increased from 0.016 g/dL on day 1 to 0.65 g/dL on day 9. The specimen was from a patient with diabetes and was positive for Candida albicans. Urine glucose concentrations dropped from >2 g/dL on day l to none detected on day 9. In vitro production of ethanol in some urine specimens was also demonstrated by Saady et al. Fourteen random urine specimens testing negative for ethanol and containing variable amounts of glucose were stored at room temperature for up to 3 weeks. Five specimens produced ethanol at concentrations ranging from 0.036 to 2.327 g/dL of ethanol. In each cases yeast was identified in the urine specimen.

Conversely, in the 6 glucose positive specimens in whom no yeast was found, no in vitro production of ethanol was observed. Jones et al. found that the production of ethanol by C. Albicans at room temperature could be prevented by the addition of sodium fluoride at a strength of 1 or 2% (w/v), but not at concentrations of 0.75% (w/v) or less. Storing the urine at 4 °C also prevents the production of ethanol in urine by C. albicans.

Ethanol Summary

A large amount of literature data exists indicating that under certain storage conditions, ethanol can be produced in blood in vitro. These conditions include higher temperatures, contamination with certain microorganisms, and the absence of chemical preservatives. When blood is preserved with fluoride, the ethanol concentration remains essentially unchanged for short periods of time, regardless of the storage temperature.

Long term storage of fluoridated blood samples will usually cause decreases in ethanol concentration. Ethanol is less likely to be produced in urine specimens, except in the rare instance of high glucose concentrations and certain microorganisms. When changes in ethanol concentration are observed, they are usually decreases in concentration. Changes in ethanol concentration in biological fluids can be minimized if the specimens are preserved with sodium fluoride and stored at as low a temperature as possible.

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