ISOTACHOPHORETIC DETECTION OF MICROBIAL METABOLITES ASSOCIATED WITH ETHANOL PRODUCTION IN URINE

* *D. R. Harper, Ph.D. ; and P. D. Martin, B. Tech.

SYNOPSISCapillary isotachophoresis has been used to determine

quantitatively the organic acid metabolites obtained when a strain of Escherichia coli is cultured in urine. The method gives a good separation of anionic species and is fast and reliable. In addition, microliter amounts of urine can be analyzed without prior sample preparation. Experiments with and without glucose as a fermentable substrate indicate that this is essential for the production of ethanol byE. coli in urine.

The authors conclude that unless glucose is present and the organic acid metabolites are detected any ethanol found in the urine could not have been produced by microbial action.

INTRODUCTIONUrine samples are received in forensic science

laboratories from suspected drunken driving cases and as post-mortem samples to estimate the level of ethanol present. If these urine samples become contaminated with microorganisms either in vivo or in vitro and a suitable substrate is present, ethanol may be produced microbiologically, and the subsequent assay will not truly reflect the quantity of ethanol previously imbibed. Some blood samples taken from cadavers have been shown to contain high levels of ethanol although it is known that the deceased did not imbibe any alcoholic beverage before death. Similar findings have been observed in post-mortem urine samples (Corry, 1980). In such cases, bacteria, yeasts, and less often moulds can usually be isolated which have the capability of producing ethanol from glucose.

All urine samples from normal healthy people contain some carboydrate material which may range from simple sugars to more complex glycoproteins. Glucose is found in the urine and the concentration can be substantial: forexample, if a person is suffering from alimentary or renal

*

Biology Research, Metropolitan Police Forensic Science Laboratory, 109 Lambeth Road, London, England SE1 7LP, UNITED KINGDOM.

609

glycosuria; or has had long periods of renal or hepatic disease; or is pregnant or diabetic; or is subjected to emotional or mental stress. Urino-genital tract infections can result in the presence of large numbers of bacteria or yeasts in the urine; when this is combined with the presence of a high sugar level, ethanol produced by the microbes cannot readily be distinguished from that excreted by the donor of the urine.

In order to prevent contamination occurring after the sample has been taken, sodium fluoride is used as a preservative in certain samples received for forensic investigation. Because this normally prevents microbial growth, it often makes impossible the isolation and enumeration of organisms. Therefore, a method must be found which will show that microbial activity had taken place and, consequently, that the ethanol level of the sample could have been affected.

During microbial growth a number of different metabolic pathways can be followed; these depend not only on the type of organism but also upon the substrate that is utilized. The bacterium chosen for this study was E. coli because it is responsible for about 80% of urino-genital infections and is, therefore, likely to be encountered as a contaminant in urine samples received in the laboratory for analysis.

E. coli converts glucose to ethanol via a "mixed-acid" fermentation (Figure 1) from which some low molecular weight carboxylic acids should be detectable as the acid products of metabolism. Therefore, a series of experiments was designed to determine whether E. coli can produce ethanol when grown in urine, and whether the detection of carboxylic acids can be used as confirmation of microbial ethanol production. These organic acids are detected by gas chromatography, high performance liquid chromatography, and enzymic assay. Moreover, Bocek et al. (1978) and van der Hoeven and Franken (1980) have described methods whereby isotachophoresis is used for the rapid detection of a variety of low molecular weight carboxylic acids, from small samples, without any prior sample treatment.

This study was concerned with the quantitative estimation by isotachophoresis of pyruvate, lactate, and acetate ions.Isotachophoresis

Isotachophoresis is a method for the separation of ionic species that have the same charge sign (in the experiments detailed below only the anionic species were detected) but, unlike common electrophoretic techniques, all

610

ions examined have the same migration velocity. Separation occurs because most of the ionic species have different effective mobilities which are dependent on charge, viscosity, molecular size and shape, solvation, and temperature. The sample ions are separated in a narrow bore, Teflon capillary tube. In our system (Figure 2) the sample ions are sandwiched between leading and terminating electrolytes (Figure 3a). The leading electrolyte comprises a single anionic species with an effective mobility higher than any of the sample ions and cations which must have a buffering capacity at the pH at which separation of the sample ions takes place. The anionic species present in the terminating electrolyte has an effective mobility which is lower than any of the sample ions being investigated.

A constant current is used to achieve separation. Once steady-state is reached— this will depend on the current and the length of the capillary tube— all of the ions migrate with the same velocity even though their effective mobilities are different. Each separate ionic species will be in a discrete zone, and the zones will be arranged in order of their decreasing effective mobilities. Mixed zones may arise which are due either to different ions with the same effective mobilities or to the absence of steady-state conditions. The electric field strength (volts/cm) from leading to terminating zones rises (Figure 3b) and because the current is constant, the conductivity drops, accordingly. Therefore, the changes in conductivity within each zone, measured with a conductivity meter, will be diagnostic for the particular ions within that zone (Figure 4).

MATERIALS AND METHODSMicrobiology

Urine was sterilized by filtration (Sterifil Aseptic Filtration System, 0.45 um pore size; Millipore-UK-Ltd., London) and dispensed aseptically into screw-capped bottles. A glucose solution was filter-sterilized and aseptically added when required, to give a final concentration in theurine of 0.5% (w/v). Bottles (175 ml) containing 150 ml ofculture were incubated at 37°C; screwcaps were tight. Inocula were 0.1 ml portions of exponentially growing cultures of E. coli (NCTC 10538) in 10 ml of the appropriate medium in universal bottles.

At intervals during growth, well-mixed portions of culture were removed and the number of viable cells wasdetermined by plating the sample onto tryptone-soya agar (Oxoid Ltd.) using a "Spiral System" (Spiral Systems

611

Marketing; Bethesda, MD, USA). Duplicate samples were plated at all times; incubation was aerobic at 30°C overnight.

Further samples were taken and treated as follows: Approximately 1 ml was filter-sterilized and stored at 4°C in a sterile Bijou bottle for the determination of ethanol by gas chromatography. Approximately 0.5 ml was filter-sterilized and stored as described above, for the determination of organic acids by isotachophoresis.Ethanol Determination

Two Perkin Elmer F45 gas chromatographs equipped with flame ionisation detectors were used for duplicate analyses of the samples. One instrument was fitted with a column of 0.2% (w/v) polyethylene glycol 1500 on Mesh 60 - 80 Carbopak C, the other with a column of 10% (w/v) polyethylene glycol400 on Mesh 100 - 120 Chromosorb W. An internal standard, n-propanol, was added to each sample. Bottles were attemperated at 60°C before head-space vapor was injected automatically onto the columns.Organic Acid Determination

The instrument used to detect these metabolites was an LKB 2127 analytical tachophor equipped with a 230-mm Teflon capillary tube (internal diameter: 0.5 mm) and an integral conductivity meter. The instrument was also fitted with an ultra-violet detector but this was not used for these estimations. Samples of 2 ul were directly injected into the capillary tube (Figure 2).

The chart speed was maintained at 10 mm/minute with a 200 mV input to the recorder.

The electrolyte system used was:Leading electrolyte5mM hydrochloric acid in 0.25% (v/v) TritonX-100, adjusted to pH 3.9 with gamma-aminobutyric acid.Terminating electrolyte5 mM heptanoic acid adjusted to pH 5 with sodium hydroxide.

Aspartic acid was added to each sample in a ratio of 1 : 1 as an internal standard.

612

Acids in the urine were quantitated by relating zone length to concentration. To assess the reproducibility of the isotachophoretic technique we prepared a standard curve for each compound on 2 separate occasions.

All separations were performed with a constant current of 100 uA.

RESULTS AND DISCUSSIONTo achieve the best resolution of the carboxylic acids,

we had to determine the optimal separation conditions for isotachophoresis. Therefore, we investigated various electrolyte systems before a final choice was made. We achieved clear and rapid resolution with the system described and used it throughout the investigation.

During research with different electrolyte systems we observed that, for practical purposes, optimal theoretical conditions did not always produce the best isotachophoregrams. For instance, according to the theory of isotachophoresis, the pH and concentration of the terminating electrolyte should be chosen to give as many unit charges as those in the leading electrolyte. Since the pKa of heptanoic acid is 4.89, it is 50% dissociated at this pH whereas the HC1 is 100% dissociated. Thus, one would have expected that optimal conditions are achieved when the molarity of the leading electrolyte is half that of the terminating electrolyte. However, in practice we achieved the best separations when the molarities of the leading and terminating electrolytes were the same. Therefore, although theoretical considerations should be heeded, the choice and concentration of the electrolytes is largely determined by experimentation.

Pyruvic acid, lactic acid, and acetic acid were resolved and determined quantitatively in the presence of the internal standard, aspartic acid.

The internal standard originally used in this study was iso-butyric acid, but it could not be resolved adequately from either n-butyric or propionic acids. Although this is not pertinent with regard to the estimation of metabolites of E. coli, it could prove troublesome in future work when investigating the metabolic products of other microorga­nisms. Aspartic acid was chosen because of its pK value and its point of separation, and because it is not detected in the growth medium following the proliferation of E. coli (NCTC 10538).

613

A linear relationship was found between the concentra­tions of the 3 carboxylic acids which were assayed and their zone lengths (Figures 5, 6, and 7); concentrations up to and including 40 mM were examined. The isotachopho-regram of an aqueous solution showed a clear separation of pyruvic, lactic, and acetic acids and the standard, aspactic acid (See Figure 8).

A typical analytical trace of an E. coli (NCTC 10538) culture in urine which contained 0.5% (w/v) glucoseindicated that pyruvic acid, lactic acid, and acetic acid were produced (Figure 9) . The presence of glucose did not affect the number of viable organisms (Figure 10). However, ethanol was produced only when glucose had been added to the urine. The 3 carboxylic acids were detected when glucose was present, but in its absence only a small amount of acetic acid was excreted by the bacteria. (Hydrolysis of acetyl-S-CoA would account for this.)

In the urine which contained glucose, E. coli (NCTC 10538) produced more than 2.5 mM ethanol, 6.9 mM aceticacid, and 10.5 mM lactic acid within 60 hours (Figure 11). In contrast to the increase in concentrations of these metabolites throughout the incubation period, the level of pyruvic acid reached a maximum at about 20 hours and then decreased. This is most likely due to the utilization of this compound, a key metabolite in carbohydrate breakdown, during subsequent metabolic processes. The relationship between the levels of the metabolites is far fromstoichiometric. Production occurs during different periods of growth, and microbial metabolism is greatly affected by the prevailing environmental conditions.

The authors realize that forensic science laboratories do not normally possess a tachophor. These findings have not been presented in order that routine isotachophoresis is carried out, but rather that the results obtained give a better understanding of what is involved with themicrobiologial production of ethanol in urine. This workshould help the forensic scientist to be aware of some of the problems which may exist when routinely examining samples for their ethanol content.

CONCLUSIONS1) E. coli (NCTC 10538) does not produce ethanol

during growth in urine from a normal healthy person.2) E. coli (NCTC 10538) produces ethanol during

growth in urine to which glucose has been added.

614

3) If glucose is present in urine and ethanol is produced, pyruvic acid, lactic acid, and acetic acid are also produced.

4) The levels of the metabolites do not necessarily have a stoichiometric relationship.

5) Isotachophoresis is a useful and rapid technique for detecting and measuring the carboxylic acid metabolites produced by E. coli (NCTC 10538).

REFERENCESBocek, P., Pavelka, S., Grigelova, K., Demi, M. , and

Janak, J. (1978). Determination of lactic and acetic acids in silage extracts by analytical isotachophoresis. Journal of Chromatography, 154; 356-359.

Corry, J. E. L. (1980). Methods of assesing the effect of microbes in blood and urine in ethanol levels. In Goldberg, L. (ed.), Alcohol, Drugs, and Traffic Safety. Stockholm: Almqvist & Wiksell International.

Van der Hoeven, J. S., and Franken, H. C. M. (1980). The determination of various low molecular weight carboxylic acids in biological samples by isotachophoresis. In Adams, A., and Schotz, C. (eds.), Biochemical and Biological Applications of Isotachophoresis. Amsterdam: Elsevier.

615

2 Glucose

I4 Pyruvic Acid

CoA-SH

2 Acetyl-S-CoA 2 Formic Acid

CoA -SH 'V ^ CoA-SH

2 Lactic Acid Acetic'Acid Ethanol 2C02 2 H2

F i g u r e 1 . " M i x e d a c i d " f e r m e n t a t i o n o f E . c o l i .

©

leading electrolyte

terminatingelectrolyte

/ \ conductivity U.V.detector detector

Idrain port

injection

F i g u r e 2 . D i a g r a m m a t i c r e p r e s e n t a t i o n o f i s o t a c h o p h o r e s i s a p p a r a t u s .

616

A+B

leadingelectrolyte

0«00«090%0• 0 9 9 90 90 000 9909

terminatingelectrolyte

//

//

/

/ a b

/////

OOOooo o o 00 ooo

V . • •• • • •••

A

FieldStrength(volts/cm)

Figure 3. Separation of sample ions by capillary isotachophoresis.

’>ZDTD

° V

U.V. Absorbance

m r r

conductivity

time

Figure 4 . Isotachophoregram showing the separation of 4 sample ions sand­wiched between leading and terminating ions. The leading ion has the greatest conductivity and the terminating ions has the lowest.

617

Figure 5 . Calibration line for the quantitation of pyruvic acid. Each point is the mean of closely agreeing duplicate determinations.

Figure 6. Calibration line for the quantitation of lactic acid. Each po-nt is the mean of closely agreeing duplicate determinations.

618

acetic acid concentration (mM)

Figure 7 . Calibration line for the quantitation of acetic acid. Each point is the mean of closely agreeing duplicate determinations.

Figure 8 . Isotachophoregram of an aqueous solution of pyruvic, lactic, aspartic, and acetic acids.

619

Figure 9. Typical isotachophoregram of an E. coli culture in urine which contained 0.5% (w/v) glucose.

21JEco

£ZOJl_>CloLJ"o£=ru

time( hours)

Figure 10. Growth of E. coli in urine at 37°C and the associated produc­tion of ethanol. Each point is a mean of duplicate experiments.(No ethanol was detected in the urine without glucose.)

620

Figure 11. Metabolites produced by E. coli during growth in urine at 37°C. Each point is a mean of duplicate experiments. (No lactate or pyruvate was detected in the sample without glucose.)

621