Estimates of Non-Alcoholic Food-Derived Ethanol and Methanol Exposure in Humans

Abstract

Food-derived alcohol is almost not in question due to its low concentration. Nevertheless, could it pose a problem for some risk groups and forensic cases? To answer this, we aimed to simultaneously evaluate ethanol and methanol ingredients of a variety of non-alcoholic foods in two different countries and estimate their possible health and forensic consequences. Alcohols in foods were analyzed by headspace gas chromatography. Human average acute daily food consumptions and food-derived blood alcohol concentrations (BACs) were determined by using the data of The European Food Safety Authority Nutrition Survey. Methanol and ethanol ingredients of similar foods varied between the two cities. Most foods produce higher methanol concentrations than the maximum allowable dose level (23 mg). Especially fruit juices lead to the critical level of ethanol for children (6 mg/kg body weight). Based on the results, adult daily intake of selected food groups does not bear ethanol that exceeds the legal limit of BAC or the limit not allowed from a religious perspective and does not lead to acute alcohol toxicity. But these low levels of ethanol and methanol consumed via non-alcoholic foods for life can raise the vulnerability to chronic health problems (cancer, liver cirrhosis, Alzheimer’s disease, autism, ocular toxicity and alterations in fetal development) and may lead to positive ethanol metabolite results (e.g., ethyl glucuronide) when a low cutoff level is used. Therefore, studies on the alcohol contents of various natural and processed non-alcoholic foods along with their effects on humans and new regulations on labeling the food products and conscious food consumption are of particular importance. It would also be important to consider unintentional alcohol consumption via non-alcoholic foods in the evaluation of clinical and forensic cases.

Introduction

Alcohol has always been a legal (in most countries) intoxicant and stimulant, which not only belongs to social customs but has also found its way into everyday consumed foods. Ethanol in foods is present due to natural fermentation of food sugar/starch or added as a solvent during food processing and production. It is commonly used in the formulation of beverages as a carrier for volatile and natural flavoring compounds.

The obligation to label the alcohol content of food products is not uniform. In Germany, all beverages containing >1.2% alcohol must be declared (Food Labeling Regulation) and fruit juices may contain up to 0.4% (v/v) ethanol equivalent to 3 g/L (1, 2). If other food in pre-packed containers contains alcohol, it must be indicated in the list of ingredients. In contrast, the labeling of compound alcoholic ingredients is not required if the final product contains <2% of these ingredients (1). Non-alcoholic beverages are defined as drinks that contain ethanol <0.3% and <0.5% (v/v) in Turkey and in the USA, respectively (3, 4).

Plants can synthesize ethanol from pyruvate in hypoxic conditions via fermentation by alcohol dehydrogenase (ADH). ADH functions in the disposal of acetaldehyde and in the synthesis of volatiles that define fruit flavor. In the food industry, fruits are held in an anaerobic environment and covered with special waxes that also decrease oxygen content. These treatments cause an increase in ethanol (also in methanol) concentrations in stored fruits and vegetables (5).

Drivers or people participating in an alcohol abstinence control program should consider the unintentional alcohol intake via foods especially for a short while (few hours) after consumption if the target is not alcohol but ethyl glucuronide (EtG), the direct non-oxidative metabolite of ethanol (6). As for women, low-dose ethanol exposure via foods or soft drinks throughout pregnancy and lactation may be responsible for learning and memory deficiencies. Fetal alcohol syndrome (FAS) is a severe disease associated with prenatal ethanol exposure and characterized by neurodevelopmental abnormalities of the central nervous system, growth retardation (typically low birth weight) and depression- and anxiety-related traits. It can be expected even after exposure to low doses of ethanol throughout the pregnancy (7–9). From a religious perspective (mostly in Muslims), ethanol with a concentration above 1% and produced by anaerobic fermentation is not accepted as halal (allowed) and it is forbidden (haram). Ethanol with a concentration of <1% and produced by natural fermentation is accepted as a preserving agent and it is halal (10).

Methanol is used as a fuel in fuel cells and particularly as an essential substance in the chemical industry. Moreover, it is found in small quantities in plants as free alcohol or as a constituent of esters in lignin and pectin, which principally occur in the shell of fruits. Methanol is produced by the hydrolysis of pectin, whereby it is present in nearly all alcoholic beverages, fruit juices and fruits. Proteolytic enzymes mainly fruit pectin methyl esterases added during food manufacturing cleave the pectin methylester groups and release methanol. Methanol can also be produced in small amounts during alcoholic fermentation (11).

Food additives such as aspartame and dimethyldicarbonate (DMDC) act as a source of methanol. DMDC increases microbiological stability and dissociates quickly to methanol and carbon dioxide upon addition. An amount of 250 mg/L DMDC in a beverage can increase the methanol content by 120 mg/L. Therefore, it is not allowed for fruit juices and nectars in Europe (12). The artificial sweetener aspartame is a methyl ester of a dipeptide consisting of aspartic acid and phenylalanine. It is rapidly broken down and is thought to release 10% methanol by weight. The acceptable daily intake (ADI) for aspartame is 40 mg/kg body weight (bw) per day, resulting in a maximum potential methanol exposure of 4 mg/kg bw (13).

Methanol is dangerous due to the formation of formaldehyde and formic acid. Visual disturbances and damage to the visual and auditory nerves, loss of appetite, abdominal pain, recurrent irritation of the eye and respiratory mucous membranes are characteristic symptoms of chronic methanol intoxication (14). A recent series of comprehensive in vitro studies have convincingly linked Alzheimer’s disease to very low concentrations of formaldehyde (15, 16). Formaldehyde, as a natural in-cell toxicant, can react with nucleic acids and proteins, triggering the pathophysiological changes found in the autistic brain (17). It is hypothesized that methanol ingested by women during pregnancy acts as a major autistic teratogen. The teratogenicity of methanol is particularly caused by the metabolite formaldehyde since its lethal effect on the embryo is nearly 1,000-fold lower (0.004 mg/mL) than either methanol or formic acid concentrations (18). In 2012, the California Office of Environmental Health Hazard Assessment (OEHHA) added methanol to a list of chemicals known to cause reproductive toxicity. Methanol itself is harmless but as defined by Monte it is a “Trojan horse” for formaldehyde, a class I carcinogen and mutagen, which can be produced within the arteries and veins, heart, brain, lungs, breast, bone and skin by class I alcohol dehydrogenase (ADH I) (19). ADH I is the only human enzyme capable of metabolizing methanol, which passes normal biological barriers (e.g., in the brain) that environmental formaldehyde itself cannot usually penetrate. Formic acid is oxidized to carbon dioxide through the action of formyl-tetrahydrofolate (THF) synthetase and formyl-THF dehydrogenase. This oxidation way is variable between species (it is twice as slow in humans and non-human primates compared to rats) due to the availability of folate, which determines the sensitivity to methanol intoxication (20). Deficiencies in THF might increase the blood level of formic acid in humans chronically exposed to methanol concentrations that are not normally toxic (5).

At the beginning of the 19th century, methanol was considered less toxic than ethanol. So, cough syrup and vanilla products contained methanol to be more inexpensive and safer. But this has resulted in a significant number of people becoming blind and die (21), revealed after several years, although exposed to a very low dose of methanol.

In this pilot study, for the first time, we aimed to find out the contents of both alcohols, ethanol and methanol, in some frequently consumed non-alcoholic foods marketed in two different countries (in Germany and Turkey) and estimate their daily consumptions and related blood concentrations to evaluate the effects of food-derived ethanol and methanol intake on human from different perspectives.

Materials and Methods

Purchase of samples

Commonly consumed foods (fruits, juices, vegetables, legumes, sauces, etc., n = 45) were purchased from the most available supermarkets in Giessen (Germany) and Ankara (Turkey).

Chemicals

All reagents used were of analytical grade. Ethanol was purchased from Baker and Merck, methanol from Sigma-Aldrich and Merck, tert-butanol and n-propanol from Merck. Ultrapure water was obtained from a MES Mp Minipure water system (Turkey) or Thermo Scientific Unity Lab Services Clinical Feed Water Systems RO ECO (Germany).

Calibrators, controls and validation studies

Pure ethanol and methanol reagents were used to prepare calibration and control solutions in distilled water. Calibration curve was linear between 0.06 and 3.84 g/L (in Giessen) or 0.24 and 3.79 g/L (in Ankara) for both ethanol and methanol with r² values of around 0.999 where five concentration levels with five replicates were studied. Three-level control samples (25, 95 and 380 mg/dL in Ankara; 30, 150 and 400 mg/dL in Giessen) were used to evaluate the method validity and stability according to international method validation guidelines (19). Since no reference matrices are not available for food types evaluated in this study, distilled water was used as a blank sample matrix in validation studies. Selectivity and specificity were evaluated by determining the lack of interfering peaks at the interested retention times in fortified (with ethanol, methanol, acetone, isopropanol and n-propanol or tert-butanol) and non-fortified blank samples. Imprecision and accuracy parameters were tested for in-day and within-days, up to 5 days with five replicates of each level. Limit of detection (LOD) and limit of quantification (LOQ) were calculated from consecutive measurements (n = 10) of the lowest control samples. Signal-to-noise ratio of the analyte response was ≥3 for LOD and ≥10 for LOQ. The required peak identification criteria were fulfilled at the LOD and acceptable precision and accuracy were achieved at the LOQ. The recovery of the method was evaluated from the results of spiked blank samples (with three different concentrations) compared to those of neat standards with 100% recovery.

Sample preparation

All samples were prepared freshly for analysis after purchase. Food samples, calibrators, or controls (200 μL or mg in Giessen, 800 μL or mg in Ankara) were added to a 20 mL headspace vial along with 100 μL (in Giessen) or 200 μL (in Ankara) of the internal standard tert-butanol (7.8 mg/dL in Giessen) or n-propanol (40 mg/dL in Ankara), then tightly closed and placed on the headspace autosampler for ethanol and methanol testing.

Instrumental analysis

A headspace gas chromatographic technique with a flame ionization detector (HS-GC--FID) was used for measuring ethanol and methanol contents of foods simultaneously. Technical instrument details are expressed in Table I.

Table I.

Parameters of Headspace Gas Chromatography

	Ankara	Giessen
Equipment	Agilent, 7890B GC and 7694E Headspace	Autosystem XL with Turbomatrix 110 HS-Sampler (Perkin Elmer)
Detector	Flame ionization detector	Flame ionization detector
Carrier gas	Helium	Nitrogen
Detector gas	Hydrogen and dry air	Hydrogen and synthetic air
Column	DB-ALC1 and DBALC2 (30 m/0.320 mm/1.80 µm, 30 m/0.320 mm/1.2 µm)	Fused silica quartz capillary column 30 m/0.53 mm Optima Wax 2.00 μm
Mode	Split (ratio 20:1)	Split (ratio 20:1)
Injection time	1 minute	0.03 minute
Injection temperature	250°C	120°C
Vial temperature	70°C	70°C
Loop temperature	80°C	80°C
Transfer line temperature	90°C	120°C
Pressure build-up time	3 minutes	1 minutes
Detector temperature	Dual detector: 260°C	250°C
Analysis time	6 minutes	2.5 minutes
Thermostatic temperature	80°C	80°C
Thermostat time	15 minutes	15 minutes

Measurements and statistical evaluation

Methanol and ethanol contents of food samples were calculated by GC (gas chromatography) software program provided by the manufacturer. MS Excel program was used for further calculations to collect data and visualize the results. Acute daily intake amounts of our selected food types were extracted from the large data of the The European Food Safety Authority (EFSA) Nutrition Survey (22) and collected under five groups such as fruits and fruits products, fruit juices, legumes, vegetables and vegetable products, and sauces and condiments. The average nutritionally acute ethanol and methanol intakes for adults with an average weight of 60 kg were estimated with a scenario consumption of both 0.5 kg (fruits, juices, legumes and vegetables/vegetable products) or 70 g (jams/marmalades and sauces/condiments) and the stated amount in the nutrition survey for each food type or group. Likewise, the peak blood alcohol concentration (BAC) produced by each food type or food group was also calculated with the use of the Widmark formula [C (g/L) = Ingested alcohol (g)/weight (kg) × volume of distribution (0.6)].

Results

Imprecision and accuracy results are expressed in Table II as the mean value of three different levels analyzed in 1 day and in 5 days consequently, where relative standard deviation (RSD) and bias were found below 15% and ±15%, respectively. Recovery was between 80% and 120% for three levels, and no interference (in a mixed and blank matrix) and no carryover (after the highest concentration) were observed. The mean ethanol and methanol contents of various foods analyzed in Giessen and Ankara were subject to strong fluctuations (Table III). Different brands and species of foods in similar categories may vary in alcohol contents between two cities or even within a city. Thus, our results may not be representative of all similar food categories. There were about 3.2 times higher values for ethanol and 1.3 times higher values for methanol in Giessen than in Ankara considering the analyzed food samples in this study. In general, methanol concentrations ranged from 0.02 to 0.75 g/kg (or g/L) in Giessen and 0.02 to 0.8 g/kg (or g/L) in Ankara, and ethanol concentrations ranged from 0.02 to 1.09 g/kg (or g/L) in Giessen and 0.02 to 0.96 g/kg (or g/L) in Ankara.

Table II.

Method Validation Results

	Ankara		Giessena
	Ethanol	Methanol	Ethanol	Methanol
Accuracy (Bias, %)	−2.72	−2.40	1.86	0.90
Imprecision (RSD, %)	1.38	1.51	0.92	1.17
LOD (g/L)	0.013	0.005	0.034	0.03
LOQ (g/L)	0.045	0.016	0.121	0.12
Linearity range (g/L)	0.12–3.79	0.12–3.79	0.06–3.84	0.06–3.84

a

From validation 2011/2018 using Valistat 2.0.

Table III.

Ethanol and Methanol Contents of Selected Foods and Estimated Peak BAC After 0.5 kg Intake of Fruits, Fruit Juices, Legumes and Vegetables/Vegetable Products, and 70 g Intake of Fruit Products (Jam or Marmalades) and Sauces/Condiments

Foods		Alcohol contenta				BAC (mg/dL)
		Ethanol		Methanol		Ethanol		Methanol
		Ankara	Giessen	Ankara	Giessen	Ankara	Giessen	Ankara	Giessen
Fruits and fruit products (g/kg)	Pear-1	0.09	0.08	0.14	0.18	0.13	0.11	0.19	0.25
	Pear-2	b	0.57	b	0.55	b	0.79	b	0.76
Mean ethanol: Mean methanol:	Apple	<LOD	0.1	0.05	0.09	b	0.14	0.07	0.13
0.04 (Ankara) 0.05 (Ankara)	Raspberry	0.02	0.42	0.02	0.28	0.03	0.58	0.03	0.39
0.25 (Giessen) 0.13 (Giessen)	Strawberry	0.07	0.02	0.03	0.08	0.10	0.03	0.04	0.11
	Apricot	0.03	0.04	0.04	0.06	0.04	0.06	0.06	0.08
	Blackberry	0.02	0.3	0.02	0.14	0.03	0.42	0.03	0.19
	Blueberry	b	0.37	b	0.13	b	0.51	b	0.18
	Apricot jam	<LOD	<LOD	0.03	0.04	b	b	0.01	0.01
	Strawberry jam	<LOD	<LOD	0.03	0.03	b	b	0.01	0.01
	Cherry jam-1	0.02	0.31	0.06	0.16	0.004	0.06	0.01	0.03
	Cherry jam-2	<LOD	<LOD	0.05	0.05	b	b	0.01	0.01
	Orange marmalade	c	<LOD	c	0.05	b	b	b	0.01
	Hawthorn marmalade	c	<LOD	c	0.06	b	b	b	0.01
	Red currant jam	b	<LOD	b	0.06	b	b	b	0.01
	Rose jam	<LOD	<LOD	0.02	<LOD	b	b	0.004	b
Fruit juices (g/L)	Peach juice-1	<LOD	0.1	0.06	0.04	b	0.14	0.08	0.06
	Peach juice-2	<LOD	0.26	0.06	0.06	b	0.36	0.08	0.08
Mean ethanol: Mean methanol:	Apricot juice-1	0.03	0.04	0.05	0.04	0.04	0.06	0.07	0.06
0.16 (Ankara), 0.09 (Ankara)	Apricot juice-2	0.25	0.26	0.44	0.03	0.35	0.36	0.61	0.04
0.21 (Giessen) 0.06 (Giessen)	Apple-cherry juice	<LOD	0.16	0.03	0.03	b	0.22	0.04	0.04
	Apple juice-1	0.04	0.3	0.03	0.08	0.06	0.42	0.04	0.11
	Apple juice (Bio)-2	0.35	0.3	0.07	0.06	0.49	0.42	0.10	0.08
	Cherry juice	<LOD	0.42	0.03	0.13	b	0.58	0.04	0.18
	Watermelon juice (natural)	0.12	0.04	0.05	0.07	0.17	0.06	0.07	0.10
Legumes (g/kg)	Canned pea	0.2	0.62	0.15	0.13	0.28	0.86	0.21	0.18
	Canned maize	0.79	1.09	0.1	0.08	1.10	1.51	0.14	0.11
Mean ethanol: Mean methanol:	Canned green beans	0.02	0.06	0.8	0.21	0.03	0.08	1.11	0.29
0.22 (Ankara), 0.39 (Ankara)	Canned kidney beans	0.03	<LOD	0.1	0.1	0.04	b	0.14	0.14
0.59 (Giessen) 0.13 (Giessen)	Canned white beans	0.05	<LOD	0.8	0.12	0.07	b	1.11	0.17
Vegetables and vegetable products (g/kg)	Canned tomatoes	0.03	0.04	0.25	0.1	0.04	0.06	0.35	0.14
	Canned tomatoes past	0.2	<LOD	0.15	0.08	0.28	b	0.21	0.11
Mean ethanol: Mean methanol:	Canned tomatoes sauce	0.14	<LOD	0.22	0.11	0.19	b	0.31	0.15
0.31 (Ankara), 0.15 (Ankara)	Canned roasted eggplant	0.03	0.05	0.16	0.16	0.04	0.07	0.22	0.22
0.09 (Giessen) 0.15 (Giessen)	Tomato-1	0.03	<LOD	<LOD	0.28	0.04	b	b	0.39
	Tomato-2 (cherry)	b	0.1	b	0.34	b	0.14	b	0.47
	Pickle-1	0.79	0.14	0.05	0.06	1.10	0.19	0.07	0.08
	Pickle-2	0.96	0.13	0.06	0.06	1.33	0.18	0.08	0.08
Sauces and condiments (g/kg)	Hot spice pasta	0.1	0.47	0.19	0.33	0.02	0.09	0.04	0.06
	Hot chili sauce	<LOD	0.67	0.07	0.14	b	0.13	0.01	0.03
Mean ethanol: Mean methanol:	Spice paste (Würzpasta)	0.46	0.7	0.43	0.75	0.09	0.14	0.08	0.15
0.17 (Ankara) 0.14 (Ankara)	Ketchup-1	0.11	0.19	0.07	0.09	0.02	0.04	0.01	0.02
0.34 (Giessen) 0.27 (Giessen)	Ketchup-2	0.09	0.13	0.03	0.02	0.02	0.03	0.01	0.004
	Mayonnaise-1	<LOD	0.15	0.05	<LOD	b	0.03	0.01	b
	Mayonnaise-2	0.09	0.05	<LOD	<LOD	0.02	0.01	b	b

a

Three replicate analysis have been performed per sample and mean value has been given;

b

No data;

c

Very high values due to long storage time (about 1 year).

In Giessen, the highest mean methanol was found in spice paste but in canned foods, mostly beans in Ankara. Canned maize showed the highest ethanol content (1.09 g/L) in Giessen followed by spice paste and chili sauce group (mean: 0.61 g/L). Pickles had the highest mean ethanol content in Ankara (mean: 0.88 g/L).

The mean methanol and ethanol contents of fresh fruits and jams (0.13 and 0.25 g/kg, respectively) were higher than fruit juices (0.06 and 0.21 g/L, respectively) in Giessen. With the amounts of 0.55 g/kg methanol and 0.57 g/kg ethanol pear (pear-2) had the highest alcohol content among fruits. Fresh tomato (cherry) came next with the highest value of methanol (0.34 g/kg), and canned pea (0.62 g/kg), raspberry and cherry juice (0.42 g/kg) with the highest value of ethanol. Fresh fruits and fruit products (jams) showed six times more ethanol and about three times more methanol content in Giessen than in Ankara. Fruit juices, in Giessen, contained higher ethanol and lower methanol than Ankara. Canned legumes showed higher methanol and pickles showed higher ethanol content in Ankara than in Giessen. Estimated blood concentrations of ethanol and methanol after 0.5 kg or 70 g of food intake by an adult weighing 60 kg are expressed in Table III. None of the fruit juices analyzed did exceed the legally allowed blood ethanol limits (3–5 g/L). With a scenario consumption of 70 g of spice paste, the highest concentration of methanol (0.75 g/kg in Giessen) may reach to 0.15 mg/dL in blood. Canned tomatoes (0.25 g/kg methanol in Ankara) or fresh cherry tomatoes (0.34 g/kg in Giessen) may result in 0.35 or 0.47 mg/dL methanol in blood after 0.5 kg intake. Around 400 mg methanol might be incorporated if 0.5 kg canned bean (methanol concentration: 0.8 g/kg) is ingested, and around 300 mg methanol might be taken if 0.5 kg pear (methanol concentration: 0.55 g/kg) is ingested producing methanol blood levels as 1.11 and 0.76 mg/dL, respectively. Drinking in an amount of 0.5 L apricot juice (methanol concentration 0.44 g/L in Ankara) can lead to 0.61 mg/dL blood methanol level. Apple juice samples showed methanol concentrations between 0.03 and 0.08 g/L, corresponding 15 and 40 mg methanol if 0.5 L consumed.

Nutritionally acute exposure levels of ethanol and methanol for both cities were estimated by using the daily food intake data obtained from EFSA Nutrition Survey. The results are presented under five different food groups like fruits and fruit products, fruit juices, legumes, vegetable and vegetable products, and sauces and condiments along with their corresponding predicted peak BAC values (Tables IV and V) and levels for per bw (Tables VI and VII).

Table IV.

Daily Acute Ethanol Consumption and Estimated Peak BAC Values of Food Groups

Foods	Average food intake for adults (EFSA Survey data)			Ethanol content (g/L or kg)		Ethanol consumption
	g/day	SD	99th percentile (g/day)	Ankara	Giessen	g/day				BAC mg/dL				99th percentile (g/day)		99th percentile BAC (mg/dL)
						Ankara	SD	Giessen	SD	Ankara	SD	Giessen	SD	Ankara	Giessen	Ankara	Giessen
Fruits and fruits products	154.96	173.53	684.67	0.040	0.240	0.006	0.007	0.037	0.042	0.017	0.019	0.103	0.116	0.027	0.164	0.076	0.456
Fruit juices	972.64	877.57	4,500.00	0.160	0.210	0.156	0.140	0.204	0.184	0.432	0.390	0.567	0.512	0.720	0.945	2.000	2.625
Legumes	46.88	47.84	187.87	0.220	0.59	0.010	0.011	0.028	0.028	0.029	0.029	0.077	0.078	0.041	0.111	0.115	0.308
Vegetables and vegetable products	85.72	71.4	284.4	0.310	0.090	0.027	0.022	0.008	0.006	0.074	0.061	0.021	0.018	0.088	0.026	0.245	0.071
Sauces and Condiments	79	74.56	336.82	0.170	0.300	0.013	0.013	0.024	0.022	0.037	0.035	0.066	0.062	0.057	0.101	0.159	0.281

Table V.

Daily Acute Methanol Consumption and Estimated Peak BAC Values of Food Groups

Foods	Average food intake for adults (EFSA Survey data)			Methanol content (g/L or kg)		Methanol consumption
	g/day	SD	99th percentile (g/day)	Ankara	Giessen	g/day				BAC mg/dL				99th percentile (g/day)		99th percentile BAC (mg/dL)
						Ankara	SD	Giessen	SD	Ankara	SD	Giessen	SD	Ankara	Giessen	Ankara	Giessen
Fruits and fruits products	154.96	173.53	684.67	0.040	0.130	0.006	0.007	0.020	0.023	0.017	0.019	0.056	0.063	0.027	0.089	0.076	0.247
Fruit juices	972.64	877.57	4,500.00	0.090	0.060	0.088	0.079	0.058	0.053	0.243	0.219	0.162	0.146	0.405	0.270	1.125	0.750
Legumes	46.88	47.84	187.87	0.39	0.13	0.018	0.019	0.006	0.006	0.051	0.052	0.017	0.017	0.073	0.024	0.204	0.068
Vegetables and vegetable products	85.72	71.4	284.4	0.150	0.150	0.013	0.011	0.013	0.011	0.036	0.030	0.036	0.030	0.043	0.043	0.119	0.119
Sauces and Condiments	79	74.56	336.82	0.140	0.270	0.011	0.010	0.021	0.020	0.031	0.029	0.059	0.056	0.047	0.091	0.131	0.253

Table VI.

Daily Acute Ethanol Exposure via Non-Alcoholic Food Consumption

Foods	Average food intake for adults (EFSA Survey data)			Ethanol content (g/L or g/kg)		Ethanol consumption
	g/day	SD	99th percentile	Ankara	Giessen	g/day				mg/kg bw day for 60 kg				99th percentile (g/day)		99th percentile (mg/kg bw day)
						Ankara	SD	Giessen	SD	Ankara	SD	Giessen	SD	Ankara	Giessen	Ankara	Giessen
Fruits and fruits products	154.96	173.53	684.67	0.040	0.240	0.006	0.007	0.037	0.042	0.103	0.116	0.620	0.694	0.027	0.164	0.456	2.739
Fruit juices	972.64	877.57	4,500.00	0.160	0.210	0.156	0.140	0.204	0.184	2.594	2.340	3.404	3.071	0.720	0.945	12.000	15.750
Legumes	46.88	47.84	187.87	0.220	0.59	0.010	0.011	0.028	0.028	0.172	0.175	0.461	0.470	0.041	0.111	0.689	1.847
Vegetables and vegetable products	85.72	71.4	284.4	0.310	0.090	0.027	0.022	0.008	0.006	0.443	0.369	0.129	0.107	0.088	0.026	1.469	0.427
Sauces and condiments	79	74.56	336.82	0.170	0.300	0.013	0.013	0.024	0.022	0.224	0.211	0.395	0.373	0.057	0.101	0.954	1.684

Table VII.

Daily Acute Methanol Exposure via Non-Alcoholic Food Consumption

Foods	Average food intake for adults (EFSA Survey data)			Methanol content (g/L or g/kg)		Methanol consumption
	g/day	SD	99th percentile	Ankara	Giessen	g/day				mg/kg bw day for 60 kg				99th percentile (g/day)		99th percentile (mg/kg bw day)
						Ankara	SD	Giessen	SD	Ankara	SD	Giessen	SD	Ankara	Giessen	Ankara	Giessen
Fruits and fruits products	154.96	173.53	684.67	0.040	0.130	0.006	0.007	0.020	0.023	0.103	0.116	0.336	0.376	0.027	0.089	0.456	1.483
Fruit juices	972.64	877.57	4,500.00	0.090	0.060	0.088	0.079	0.058	0.053	1.459	1.316	0.973	0.878	0.405	0.270	6.750	4.500
Legumes	46.88	47.84	187.87	0.39	0.13	0.018	0.019	0.006	0.006	0.305	0.311	0.102	0.104	0.073	0.024	1.221	0.407
Vegetables and vegetable products	85.72	71.4	284.4	0.150	0.150	0.013	0.011	0.013	0.011	0.214	0.179	0.214	0.179	0.043	0.043	0.711	0.711
Sauces and condiments	79	74.56	336.82	0.140	0.270	0.011	0.010	0.021	0.020	0.184	0.174	0.356	0.336	0.047	0.091	0.786	1.516

Discussion

Similar results are found by Lund et al. (23). They determined ethanol levels between 90 and 100 ppm and methanol levels between 10 and 80 ppm in citrus products, and ∼140 mg/L (12–640 mg/L) methanol in non-alcoholic fruit juices. We found about 60 (30–130 in Ankara) and 90 (30–440 in Giessen) mg/L methanol, which is almost equal to the average level in white wine or within the range of levels found for red wine (24).

Methanol has been detected in various fruits and vegetables like roasted filberts, carrots, onions, Brussels sprouts, celery, peas, parsnips and potatoes and the concentrations ranged from 1.5 to 7.9 mg/kg in beans, lentils and split peas (25). We detected methanol content as 150 mg/kg for vegetables (tomato, eggplant and pickle) and 130 (Giessen)—390 (Ankara) mg/kg for legumes (beans, maize and pea) where these high contents may result due to the industrial processing of the foods.

The consumption of 0.5 kg of some selected foods shows that most of them produce higher concentrations of methanol than the maximum allowable dose level (23 mg methanol per day for ingestion) indicated by the OEHHA (26). According to the European (EU) standards, methanol content is allowable up to 4,000 mg/L in spirits with 40% v/v ethanol concentration (27). But there is no legislation or standards for homemade alcoholic beverages and for non-alcoholic beverages like fruit juices and other food products. Methanol released from food-derived pectin consumed daily in an amount of 5 g, which corresponds to ∼500 g of fruits and vegetables consumed per day, may play a part in the development of non-alcoholic cirrhosis in the liver (7, 28). Total exposure to methanol from natural sources is uncertain but intakes of 10.7 and 33 mg per day have been reported in US consumers (29). Estimated human methanol production from fruits and vegetables is ∼1,000 mg every day (30). In general, a normal human body generates 0.3–0.6 g/day methanol (27). The minimum lethal dose of methanol is 0.3–1 g/kg bw (20–60 g or 25–75 mL/person in a 60 kg adult) and the minimum dose associated with ocular toxicity is around 10 mL (8 g or 133 mg/kg bw) (31). As little as 15 mL of methanol reportedly has caused blindness, and several ounces (70–100 mL) may be fatal (32). Several studies detected mean methanol blood concentrations of 0.17 and 0.26 mg/dL after 24-hour restricted alcohol intake (33). Blood methanol level was reported 10 mg/L after eating 0.75 kg of peaches and/or apples and eating 1 kg of apples released 0.5 g of methanol that was two times over the normal production (28). We estimated maximum blood methanol concentration as 1.11 mg/dL in Ankara and 0.76 mg/dL in Giessen after consuming 0.5 kg of canned beans and one kind of pear, respectively, where these values exceed the normal endogen blood methanol concentration but lie under the treatment threshold (20 mg/dL) (34, 35).

According to the Nutrition Survey data, the most consumed food type by an adult was fruit juices. We calculated human average daily methanol production and found about 58 (Giessen)—88 (Ankara) mg but 270 (Giessen)—405 (Ankara) mg in 99th percentile, being within the daily production range (0.3–0.6 g/day). We calculated daily food-derived methanol intakes per bw/day and found between 0.1 and 1.46 in Ankara and 0.1 and 0.97 mg/kg in Giessen, where the highest values were observed in fruit juice group and rose to 6.75 mg/kg (Ankara) and 4.5 mg/kg (Giessen) in 99th percentile. Among aspartame users (34 mg aspartame/kg bw/day), the 99th percentile intake of methanol was about 2.4–3.7 mg/kg bw/day (29). No observed adverse effect level of methanol for humans was reported as 71–84 mg/kg bw/day and recommended an ADI between 7.1 and 8.4 mg/kg bw/day (24). Our estimated results for acute consumption of selected foods showed no risk of acute adverse effect for a healthy adult.

With the scenario consumption of 0.5 kg of canned maize (1.09 g/kg ethanol) or pickle (0.96 g/kg ethanol) 0.55 and 0.48 g ethanol, respectively, might be incorporated to the body leading to an estimated peak blood ethanol level between 0.10 and 0.11 g/L for an adult having 5 L blood volume, where this value is above or close to the acceptable legal limit for drivers in some countries, including China, Turkmenistan, Nepal, Vietnam, Iran, Hungary, Romania, Switzerland and Cuba, but under the legal limit in most European countries and the USA (36). But if Widmark formula is used for BAC estimation, up to 1.52 mg/dL blood ethanol might be observed in a 60 kg weighing person after eating of 0.5 kg of the same foods, where this value does not lead to any legal sanctions.

Based on a report, acute ethanol exposure via fruit juices (ethanol concentrations between 0.3–1.8 g/L) can be between 12.5 and 23.3 mg/kg bw per day, if preschool children (about 20 kg bw) consume 162 mL fruit juice daily, where this value exceeds the critical level of 6.0 mg/kg bw suggested for herbal medicinal products (37–39). The Committee for Human Medicinal Products (CHMP) proposes a 1.5 g maximum ethanol dose for a 20 kg weighing child with a minimum dose interval of 4 hour (40). Furthermore, the potentially median lethal dose of ethanol for all mammals is 7 g/kg bw but 3 g/kg in children (12). The human body production of ethanol per day is up to 30 g (28). Based on the EFSA Nutrition Survey, fruit juices in our study lead to 12 (in Ankara) and 1,575 mg/kg bw/ethanol in the 99th percentile. None of our evaluated foods showed acute health-threatening concentration for healthy adults, but depending on consumption dose children should be protected from potential risks of food-derived alcohol. We found the highest ethanol in pear (0.57 g/kg), cherry juice (0.42 g/L) and apple juice (0.35 g/L) respectively in fruits and fruit juices group, and if 0.5 kg (or L) of those are ingested, 0.29, 0.21 and 0.18 g ethanol might be incorporated, being above the risk level (6.0 mg/kg bw) for children. Among all foods we analyzed, canned maize (1.09 g/kg), pickle (0.96 g/kg) and hot spice paste (0.7 g/kg) had the highest ethanol contents, which are frequently consumed as or in fast foods, also might cause several risks for children. As reported by the European Medical Agency, blood ethanol levels in the range of 1–100 mg/dL in children may have adverse effects on the central nervous system. One of the reasons for being more vulnerable to alcohol toxicity of children is the low activity of ADH, which reaches the normal adult range only after 5 years of age (41).

CHMP indicated that amounts increasing blood ethanol levels up to 1.5 mg/L might be accepted, where this value was based on the fact that ethanol is an endogenous substance likely produced in the intestine. Ethanol production by microbial fermentation in the large intestine is about 3 g per day (42). Furthermore, endogenous ethanol reaches low concentrations of 0.39 ± 0.45 μg/L in the blood of sober people (38). Ethanol is metabolized to acetaldehyde and then to acetate through NAD⁺-dependent enzymes alcohol and acetaldehyde dehydrogenases respectively, and both of these enzymes show extensive polymorphism among racial groups. Rapid formation or reduced oxidation of acetaldehyde in these populations leads to higher acetaldehyde concentration in the blood, which produces the acetaldehyde syndrome (headache, vomiting, sweating, thirst, vertigo, blurred vision, confusion and respiratory difficulties). Thus, individuals predisposed to ethanol sensitivity should care for ethanol consumption in any form.

Musshoff et al. evaluated direct metabolites of ethanol after the consumption of various beverages and foods (6). No positive EtG or ethyl sulfate (EtS) were observed after drinking 1.1 and 2.0 L apple juice with a concentration of 0.3 g/L ethanol (similar concentration to our findings), which represents an ethanol dose of 0.3–0.6 g. After an intake of 1.5–2.0 L grape juice (ethanol dose: 0.9–2.5 g), EtS concentrations were detectable up to 35 hours, with negative EtG results. After taking a lot of non-alcoholic beer (3.6 g/L ethanol), sauerkraut (2 g/kg ethanol) and matured bananas (5 g/kg ethanol), EtG was found positive in urine up to 13 hours, 5 hours and 3.5 hours, respectively, when used the cutoff level as 0.1 mg/L. The authors suggested that positive EtG concentrations can occur due to foods for a limited time (24 hours).

Considering several reports (18–20), it is communicable that even low-dose ethanol intake during pregnancy via non-alcoholic foods (e.g., canned maize, pickle, spice products and fruit juices) may induce the alterations to brain development in fetal life and FAS, where the presence of risk factors for FAS, such as ethnicity, alcohol metabolism, nutrition pattern and socioeconomic status, should also be considered. In a study, animals were treated with 5% ethanol during pregnancy and lactation and indicated that prenatal ethanol exposure increased anxiety-like behavior via partially sex-dependent alteration of the actions of the brain renin–angiotensin system to impair the memory (18).

Few studies exist related to the toxicity of chronic low-dose ethanol exposure. Well-nourished rats exposed for 1 week to ethanol (5% v/v, mean blood level: 1.43 mg% ± 1.86) presented a decrease in body weight gain and an increase in relative liver weight without important toxic effects on liver morphology or blood biochemistry (43). In another study, rats were exposed to low doses of ethanol (3% v/v, blood level: 0.82 mg ± 0.2 mmol/L) for 2 months (44). Their liver mitochondria were isolated and biochemically evaluated. The results showed a significant increase in lipid peroxidation and content of oxidized proteins. They claimed that the slight mitochondrial changes due to chronic exposure to low doses of ethanol might reflect the initiation of a more diffuse injury.

Taking together, ethanol and methanol are both primarily metabolized by ADH to acetaldehyde and formaldehyde, respectively, and cleared at a constant rate regardless of the dose. The clearance of methanol is slow, particularly when compared to ethanol because ethanol is preferentially metabolized due to its greater affinity for the enzyme (about 20:1). Thus, the rate of methanol oxidation is ∼3% of the rate of ethanol (5). Naturally occurring methanol is always accompanied by ethanol, which has a preventive effect on toxicity, whereas the methanol released from additives such as aspartame would not be accompanied by ethanol and thus might cause potentially adverse effects on health.

Conclusion

To the best of our knowledge, this preliminary study is the first indicating both methanol and ethanol concentrations simultaneously in frequently consumed non-alcoholic foods available in two different countries (Turkey (Ankara) and Germany (Giessen)) and discussing their possible health and forensic consequences for humans on the basis of estimated acute daily consumption amounts and produced blood concentrations. Dietary exposure to methanol and ethanol arises from a variety of sources, and the content of these chemicals in commercially available foods depends on the nature of the food and differences in processing.

None of the foods we evaluated produce ethanol or methanol to pose serious acute health or legal problem to adult human in case of consuming 0.5 kg (L)/day, but even this amount is seen to have some consequences particularly for children who like to consume juices and fast foods more frequently, as well as for predisposed adults who likely consume regularly non-alcoholic foods, particularly fruits/juices, canned legumes (e.g., beans) or pickle.

Ethanol derived from natural foods or is extensively applied in food industry, and methanol primarily found in fruits as free or pectin-derived methanol and converted to toxic metabolites in the human body, are almost not in question due to their low concentration. But unaware consumption of these chemicals should be subjected to evaluation especially because of the importance for sensitive (children, pregnant women, and alcoholics under abstinence control), sick (cancer, liver disease, neurophysiological disorder, and folate deficiency) or religious people.

Few data exist about the chronic effects of low-dose methanol and ethanol on humans. To clarify and avoid potential health risks of food-derived methanol and ethanol, it is necessary to determine their exposure pattern during daily nutrition, and establishing new food labeling regulations along with conscious consumption especially for processed food sources is of particular importance. In this regard, although this work presented valuable preliminary results of a collaborated study from two countries, more advanced analytical (about the contents of alcohols in foods or beverages considered as non-alcoholic) and clinical research (about the health and forensic significance of food-derived alcohols to humans) should be performed.

Acknowledgment

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Conflict of Interest

The authors declare that they have no conflict of interest.