http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2015; 37(2): 193–201 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1009998

RESEARCH ARTICLE

Aliphatic alcohols in spirits inhibit phagocytosis by human monocytes

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00 La´szlo´ Pa´l1*, Ervin M. A´rnyas1*, Orsolya Bujdoso´1, Gergo Baranyi1, Ga´bor Ra´cz1, Ro´za A´da´ny1, Martin McKee2, and 00 1 Sa´ndor Szucs

1

Department of Preventive Medicine, Faculty of Public Health, University of Debrecen, Debrecen, Hungary and 2European Centre on Health of Societies in Transition, London School of Hygiene and Tropical Medicine, London, UK Abstract

Keywords

A large volume of alcoholic beverages containing aliphatic alcohols is consumed worldwide. Previous studies have confirmed the presence of ethanol-induced immunosuppression in heavy drinkers, thereby increasing susceptibility to infectious diseases. However, the aliphatic alcohols contained in alcoholic beverages might also impair immune cell function, thereby contributing to a further decrease in microbicidal activity. Previous research has shown that aliphatic alcohols inhibit phagocytosis by granulocytes but their effect on human monocytes has not been studied. This is important as they play a crucial role in engulfment and killing of pathogenic microorganisms and a decrease in their phagocytic activity could lead to impaired antimicrobial defence in heavy drinkers. The aim of this study was to measure monocyte phagocytosis following their treatment with those aliphatic alcohols detected in alcoholic beverages. Monocytes were separated from human peripheral blood and phagocytosis of opsonized zymosan particles by monocytes treated with ethanol and aliphatic alcohols individually and in combination was determined. It was shown that these alcohols could suppress the phagocytic activity of monocytes in a concentration-dependent manner and when combined with ethanol, they caused a further decrease in phagocytosis. Due to their additive effects, it is possible that they may inhibit phagocytosis in a clinically meaningful way in alcoholics and episodic heavy drinkers thereby contribute to their increased susceptibility to infectious diseases. However, further research is needed to address this question.

Aliphatic alcohols, infectious diseases, unrecorded alcohol consumption, monocytes, phagocytosis

Introduction Alcoholic beverages consumed worldwide are primarily from legal sources, with production and sale subject to control by government authorities1. However, there is growing recognition of the importance of a diverse range of alcoholic products that are consumed in many parts of the world and that escape ‘‘capture’’ in routine sales statistics1. These include counterfeit, re-labelled, and smuggled alcohols, illegally produced homemade spirits, and surrogate alcohols not officially considered suitable for human consumption such as aftershaves, perfumes, and medical tinctures containing high levels of ethanol1,2. The latest Global Status Report on Alcohol and Health estimates that the total volume is equivalent to an annual adult per capita consumption of 1.5 l in 20101. This is equivalent to 24.8% of alcohol consumed globally, varying from 8.5 to 44.3%, in high- and low-income countries (50 and 35 World Health Organisation member states, respectively)1 (countries included are

*These authors share first authorship equally. 00 Address for correspondence: Dr. Sa´ndor Szucs, Department of Preventive Medicine, Faculty of Public Health, University of Debrecen, H-4012 Debrecen, P.O. Box 9, Hungary. Tel: +36 52 460 190x77152. Fax: +36 52 417 267. E-mail: [email protected]

History Received 30 October 2014 Revised 5 January 2015 Accepted 16 January 2015 Published online 18 February 2015

specified in Ref. [1, p. 349]). Both the amount consumed and the share of overall alcohol consumption have been reported to be higher than the European average in certain Central and Eastern European (CEE) countries (see Figure 1)1,3. These include Hungary, Russia, Romania, Ukraine, and the Republic of Moldova where homemade spirits are produced using traditional methods from a variety of fruits and grains. These are widely consumed as they are much cheaper than commercial counterparts4,5. Recently, toxicologists and public health researchers have expressed concern about health risks associated with consumption of these alcoholic beverages, following the discovery of a range of harmful chemicals in them such as toxic heavy metals (lead and cadmium) and metalloids (arsenic and antimony), acetaldehyde, and ethyl carbamate5–10. Furthermore, previous studies have demonstrated that many commercial alcoholic beverages including beer, wine, cognac, whiskey, rum, and fruit-based spirits contain not only ethanol but also methanol and aliphatic alcohols [(AAs), comprising more than two carbon atoms] such as the so-called higher alcohols like 1- and 2-propanol, 1- and 2-butanol, isobutanol, and isoamyl alcohol2,4,10–13. Methanol and higher alcohols are the by-products of alcoholic fermentation and their concentration in alcoholic beverages depends on particular fruit and distillation methods used2,14. Compared with ethanol,

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Figure 1. Volume of per capita annual unrecorded alcohol consumption in Europe in 2010. Data were obtained from the Global Status Report on Alcohol and Health, WHO, Geneva 2014. The volume of per capita alcohol consumption from illegal sources was adjusted for pure ethanol.

AAs have more pronounced hepatotoxic effects and it has been suggested that excessive consumption of homemade spirits may be related to high rates of premature mortality from chronic liver diseases and cirrhosis in CEE countries2,4. In addition to effects on the liver, gastrointestinal, nervous, and cardiovascular systems, acute and chronic alcohol consumption has been found to cause numerous immunotoxic effects including decreased antigen-specific T-lymphocyte activation and proliferation, changes in T-lymphocyte cytokine synthesis and B-lymphocyte immunoglobulin production, and reduced granulocyte chemotactic and phagocytic activities15–27. Consequently, alcohol abuse has been associated with increased susceptibility to bacterial and viral infections and higher morbidity and mortality from a number of infectious diseases including tuberculosis, pneumonia, human immunodeficiency virus-1, and hepatitis C infections17,26,28. Monocytes, as essential mononuclear phagocytic cells, are recruited to the site of infections after granulocytes and provide the second line of host defence against bacteria, fungi, viruses, and virally infected cells29,30. They play a pivotal role in phagocytosis and killing of invading pathogenic microorganisms, moreover monocyte-derived macrophages are the most significant cells responsible for the engulfment and clearance of apoptotic cells29,31. Defects in monocyte functions have been proposed to contribute to the impaired anti-microbial defences noted in alcoholics17,19,22,25–27. It has been reported that ethanol can suppress monocyte phagocytosis22,25,32,33. Although the consequences of the ethanol consumption are sufficient to cause concern for public health, less attention has been paid to the possible immunotoxic effects of the higher alcohols found in alcoholic beverages. We have previously shown that AAs can inhibit superoxide-anion production and phagocytosis by human granulocytes34,35. Still, it is important to know whether these alcohols can also decrease phagocytic activity of monocytes, as this could exacerbate the already-impaired anti-microbial defences in heavy drinkers. To our knowledge, this has not previously been investigated. Thus, the aim of our study was to measure monocyte phagocytosis following treatment of cells with some higher alcohols routinely

detected in alcoholic beverages, thereby providing evidence that unintended consumption of AAs may contribute to the increased susceptibility to infectious diseases seen in alcohol abusers.

Materials and methods Chemicals Ethanol, 1- and 2-propanol, 1- and 2-butanol, 2-methyl-1propanol (isobutanol), and 3-methyl-1-butanol (isoamyl alcohol) were purchased from Merck (Darmstadt, Germany). Ficoll-histopaque (1.077 and 1.119 g/ml), zymosan A particles (from Saccaromyces cerevisiae), fluorescein isothiocyanate (FITC), and 40 ,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (Stenheim, Germany). Chamber slides for phagocytosis assay (8 wells/slide) were acquired from VWR International LLC (Radnor, PA). Mouse anti-CD14 monoclonal antibody and anti-mouse IgGconjugated Dylight 594 fluorescent dye were obtained from Beckton Dickinson Biosciences (Ko¨rnye, Hungary) and Vector Laboratories (Peterborough, United Kingdom). Human AB serum was purchased from a local blood transfusion centre. All other chemicals were of analytical grade. Subjects After informed consent and with the approval of the Institutional Ethical Committee at the University of Debrecen, peripheral blood was collected into Vacutainer tubes containing EDTA (Becton-Dickinson, Cedex, France) from healthy volunteers (n ¼ 15, seven females and eight males). They were the staff members of the Department of Preventive Medicine including lecturers, researchers, PhD students, and office workers. Age of the subjects ranged from 21 to 46 years [mean ¼ 30.1 ± 9.0 year]. All subjects were non-smokers, not heavy drinkers, had normal dietary habits, and were not taking any alcohol/medications that could influence results of the experiments. Fasting blood samples were collected between 8 and 9 AM and processed

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immediately. To study the effects of each aliphatic alcohols (eight alcohols), their mixture, and their mixture containing 10 mM ethanol on phagocytosis and obtain sufficient data for statistical analysis (6 data per type of treatment, totally 10  6 series of data) 15 experiments were performed using the blood samples of 15 subjects. Therefore, blood samples were collected from the same donor four times.

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Separation of mononuclear cells Mononuclear cells were separated as described previously36,37. In brief, blood samples were mixed with an equal volume of Hanks’ balanced salt solution (HBSS, pH 7.4) and then layered atop a discontinuous Ficoll gradient (1.077 and 1.119 g/ml). The sample was then centrifuged at 400  g (20  C, 30 min) and the mononuclear cells from the top of the Ficoll layer then collected. The cells were washed twice with HBSS and their viability determined by the trypan blue exclusion test (routinely 96–98%). Mononuclear cells separated from different subjects were not combined into a single cell suspension. Each experiment was carried out with monocytes from individual donors. Preparation of FITC-labelled and opsonized zymosan particles Zymosan particles were labelled and opsonised as described previously38. In brief, the particles (108/ml) were incubated in carbonate buffer containing FITC at a final concentration of 0.01 mg/ml for 60 min at 37  C. The particles were then washed three times and opsonised for 30 min at 37  C in HBSS containing 50% human AB serum. The FITC-labelled opsonised particles (FITC-OZ) were then washed three times, re-suspended in HBSS (at 3  107/ml), and stored at 20ºC until required for use in phagocytosis assays. Phagocytosis assay Phagocytosis of the FITC-OZ was determined as described previously33,39,40. Here, 105/ml mononuclear cells in 300 ml aliquots (in HBSS 5% containing heat-inactivated human AB serum) were placed into the wells of chamber slides and the cells allowed to adhere for 30 min at room temperature. Nonadherent cells were removed by washing and the adherent cells then treated with FITC-OZ (3  106/ml, target/effector ratio ¼ 10:1) and medium containing increasing final concentrations (e.g., 0.005, 0.05, 0.5, or 5 mM) of ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, and isoamyl alcohol. Each sample was then incubated at 37  C in a 5% CO2/95% humidified air chamber for 60 min. Other sets of monocytes were incubated in a mixture containing 10 mM ethanol and each of the aliphatic alcohols at final concentrations of 0.005, 0.05, 0.5, or 5 mM. Untreated cells served as controls. The viability of monocytes was checked using the trypan blue exclusion test after the treatments and was found to be 96–98%. Following incubation, the fluorescence of noningested particles was quenched by addition of 0.2% trypan blue solution and the cells were then fixed with a 4% paraformaldehyde solution. Monocytes were identified by an indirect immunofluorescent method. First, the chamber slide cells were incubated at room temperature for 60 min in

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phosphate-buffered saline (PBS, pH 7.4) containing antiCD14 monoclonal antibody (1:50 dilution). The cells were then rinsed three times with PBS. Monocytes incubated without anti-CD14 antibody served as negative controls. All monocytes were then stained with anti-mouse IgG conjugated with Dylight 594 fluorescent dye (1:200 dilution) for 45 min. The cells were then washed four times with PBS and the monocyte nuclei were stained with DAPI. Each slide was then removed from its chamber for microscopic evaluation. The number of FITC-OZ/cells was determined with an Axioplan fluorescent microscope (Zeiss, Oberkochen, Germany) by examining 100 monocytes in randomly selected microscopic fields/slide. Each treatment was evaluated in duplicate; each slide was assessed twice /sample. From these values, the phagocytosis index (PI, average number of ingested particles/ cell) was calculated. Statistical analysis All results are presented as mean values [ ± SD] from six independent experiments. Differences in phagocytosis by untreated monocytes and cells treated with aliphatic alcohols were determined by one-way analysis of variance (ANOVA) using a Newman–Keuls post-hoc test. Phagocytic activity of monocytes from female and male donors was compared using an unpaired t-test. p-Values50.05 were considered statistically significant.

Results There was no statistically significant difference between the phagocytic activity of untreated monocytes from female (PI: 4.2 ± 0.5) and male (4.1 ± 0.6) donors. As shown in Figure 2, monocytes incubated in HBSS containing 0.005 mM (PI: 3.62), 0.05 mM (PI: 3.44), 0.5 mM (PI: 3.33), or 5.0 mM (PI: 2.99) ethanol demonstrated significantly reduced phagocytosis compared with values seen with control cells (PI: 3.86). There was a significant difference between the phagocytic activity of untreated monocytes (PI: 3.79) and cells exposed to methanol at levels of 0.005 mM (PI: 3.33), 0.05 mM (PI: 3.09), 0.5 mM (PI: 2.71), and 5.0 mM (PI: 2.33). Figure 3 shows that monocytes incubated with 1-propanol [PI: 4.15 (control), 0.005 mM (PI: 3.55), 0.05 mM (PI: 3.28), 0.5 mM (PI: 3.05), 5.0 mM (PI: 2.72)] or 2-propanol [PI: 3.98 (control), 0.005 mM (PI: 3.37), 0.05 mM (PI: 3.28), 0.5 mM (PI: 3.05), 5.0 mM (PI: 2.72)] exhibited significantly reduced phagocytic activity compared to control cells. Figure 4 illustrates that monocytes incubated with 1-butanol [0.005 mM (PI: 3.86), 0.05 mM (PI: 3.61), 0.5 mM (PI: 3.31), 5.0 mM (PI: 2.84)] or 2-butanol [0.005 mM (PI: 3.86), 0.05 mM (PI: 3.59), 0.5 mM (PI: 3.26), 5.0 mM (PI: 2.90)] ingested significantly less FITC-OZ compared with untreated cells (PI: 4.24). Figure 5 shows that phagocytosis decreased following treatment of the cells with isobutanol [PI: 4.27 (control), 0.005 mM (PI: 4.02), 0.05 mM (PI: 3.75), 0.5 mM (PI: 3.47), 5.0 mM (PI: 3.02)]. Incubation of monocytes with isoamyl alcohol [PI: 4.34 (control), (0.005 mM (PI: 4.01), 0.05 mM (PI: 3.74), 0.5 mM (PI: 3.46), 5.0 mM (PI: 3.13)] resulted in a concentration-dependent inhibition of phagocytosis. As shown in Figure 6, phagocytosis index was also

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Figure 2. Effects of ethanol and methanol on phagocytosis. Monocytes were separated from peripheral venous blood of healthy volunteers by Ficoll density gradient centrifugation. Subsequently, the cells were incubated in Hanks’ solution containing FITC-OZ and ethanol or methanol for 1 h at 37  C. Following treatment, phagocytic activities of control and treated cells were determined as described in ‘‘Materials and methods’’ section. Mean values (± SD) of six independent experiments are demonstrated. **p50.01, ***p50.001.

Figure 3. Effects of 1- and 2-propanol on phagocytosis. Monocytes were separated from peripheral venous blood of healthy volunteers by Ficoll density gradient centrifugation. Subsequently, the cells were incubated in Hanks’ solution containing FITC-OZ, and 1- or 2-propanol for 1 h at 37  C. Following treatment, phagocytic activities of control and treated cells were determined as described in the ‘‘Materials and methods’’ section. Mean values ( ± SD) of six independent experiments are shown. ***p50.001.

significantly decreased when cells were exposed to a mixture containing each aliphatic alcohol at final concentrations of 0.005–5.0 mM [PI: 3.92 (control), 0.005 mM (PI: 3.57), 0.05 mM (PI: 3.28), 0.5 mM (PI: 3.09), and 5.0 mM (PI: 2.79)]. Compared with the cells treated only with 10.0 mM ethanol (PI: 2.88), monocytes incubated with a mixture containing 10.0 mM ethanol and aliphatic alcohols [0.05 mM (PI: 2.67), 0.5 mM (PI: 2.48) 5.0 mM (PI: 2.26)] showed significantly decreased phagocytosis.

Discussion Numerous epidemiological studies have found a strong association between alcohol abuse and increased risk of

development and progression of bacterial diseases including tuberculosis and pneumonia16,17,26,28,41. Ethanol-induced defects in humoral and cellular immunity have been considered to be a primary reason for impaired anti-microbial resistance in alcoholics17,19,22,25–27. A large volume of alcoholic beverages are widely consumed in many parts of the world so exposure to the AAs they contain may also influence immune cell function and contribute to increased susceptibility to infectious diseases in alcoholics and heavy drinkers1,41–43. As monocytes play an important role in engulfment and killing of a variety of pathogens, it was reasonable to investigate whether the higher alcohols of these beverages have an impact on their phagocytic activity29,30. The present results demonstrated that AAs found in alcoholic

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Figure 4. Effects of 1- and 2-butanol on phagocytosis. Monocytes were separated from peripheral venous blood of healthy volunteers by Ficoll density gradient centrifugation. Subsequently, the cells were incubated in Hanks’ solution containing FITC-OZ, and 1- and 2-butanol for 1 h at 37  C. Following treatment, phagocytosis of control and exposed cells was determined as described in ‘‘Materials and methods’’ section. Mean values ( ± SD) of six independent experiments are presented. **p50.01, ***p50.001.

Figure 5. Effects of isobutanol and isoamyl alcohol on phagocytosis. Monocytes were separated from peripheral venous blood of healthy volunteers by Ficoll density gradient centrifugation. Subsequently, the cells were incubated in Hanks’ solution containing FITC-OZ and isobutanol or isoamyl alcohol for 1 h at 37  C. Following treatment, phagocytosis of control and exposed cells was determined as described in the ‘‘Materials and methods’’ section. Mean values ( ± SD) of six independent experiments are depicted. *p50.05, **p50.01, ***p50.001.

beverages could inhibit phagocytosis by human monocytes in a concentration-dependent manner and, when combined with ethanol, could act additively. The observed inhibition of phagocytosis cannot be explained by the cytotoxic effects of AAs because the viability of monocytes did not change during treatment. Having shown that AAs can act in this way, the next step is to determine whether they might be expected to reach sufficient concentrations in the blood of those drinking alcoholic beverages? The concentration of AAs in different types of alcoholic drinks varies considerably11–13,44–49. Beer has been found to include up to 0.18 mM (isobutanol – 1.3 g/hl) and 0.59 mM (isoamyl alcohol – 5.2 g/hl) while red wine has been found to contain up to 0.63 mM (isobutanol –

4.7 g/hl) and 3.25 mM (methanol – 10.4 g/hl)45. Therefore, even when large volumes of these types of alcoholic beverages are consumed, the blood AA concentrations would be below the levels required to inhibit phagocytosis11. However, much higher amounts of AAs have been detected in distilled spirits4,43,44–49. Previous studies have shown that legally produced spirits such as ‘‘Kirschwasser’’ (German cherry spirit) and Scotch whiskey contained, respectively, 37.2–79.7 mM and 0.66–2.09 mM methanol, 3.7–73.6 mM and 0.85–6.59 mM 1-propanol, 0.07–0.19 mM and 0.0–0.11 mM 1-butanol, 0.04–6.40 mM and 0.0–0.05 mM 2-butanol, 1.51–5.55 mM and 2.29–5.53 mM isobutanol, 4.45–19.67 mM and 2.44–3.99 mM isoamyl alcohol44. The levels in unrecorded spirits collected in Hungary and Poland

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Figure 6. Effects of ethanol and mixture of aliphatic alcohols (AAs) on phagocytosis. Monocytes were separated from peripheral venous blood of healthy volunteers by Ficoll density gradient centrifugation. Then the cells were incubated in Hanks’ solution containing FITC-OZ, mixture of AAs or ethanol and a mixture of AAs for 1 h at 37  C. Following treatment, phagocytic activities of control and treated cells were determined as described in the ‘‘Materials and methods’’ section. Mean values (± SD) of six independent experiments are shown. *p50.05, ***p50.001.

Table 1. Estimated volume of unrecorded and recorded spirits that needs to be consumed to reach minimal inhibitory concentration of aliphatic alcohols in the blood and related blood ethanol levels. Unrecorded alcoholic beverages Samples collected in Hungarya (homemade spirits)

Recorded alcoholic beverages

Samples collected in Polandb (homemade spirits)

Samples collected in Germanyc (Kirschwasser)

Samples collected in Germanyc (Scotch whiskey)

Aliphatic alcohol

Estimated volume of spirit (ml)

Related blood ethanol level (mM)

Estimated volume of spirit (ml)

Related blood ethanol level (mM)

Estimated volume of spirit (ml)

Related blood ethanol level (mM)

Estimated volume of spirit (ml)

Related blood ethanol level (mM)

Methanol 1-Propanol 1-Butanol 2-Butanol Isobutanol Isoamyl alcohol

1.4–71 9.8–113 140–1816 24–1816 25–96 5.7–28

0.3–15 2.1–24 30–383 5.0–383 5.3–20 1.2–5.9

1.0–1570 14–2103 204–3632 n.d. 10–1211 4.6–4319

0.22–331 3.0–444 43.1–766 n.d. 2.2–255 1.0–911

3.1–6.6 3.3–66 1289–3500 38–6125 44–162 13–55

0.7–1.4 0.7–14 272–738 8.1–1292 9.3–34 2.6–12

117–371 37–288 n.d. n.d. 44–107 61–100

25–78 7.8–61 n.d. n.d. 9.3–23 13.0–21

The volume of recorded and unrecorded alcoholic beverages and concentrations of AAs were estimated by Widmark’s equation. Both are described in the Discussion. AAs, aliphatic alcohols; n.d., no data. a The concentration of AAs in distilled spirits is taken from previous research, see Ref.4. b The concentration of AAs in distilled spirits is taken from previous research, see Ref.46. c The concentration of AAs in distilled spirits is taken from previous research, see Ref.44.

were as follows: 3.43–164.2 mM methanol, 2.16–25.0 mM 1-propanol, 0.13–1.75 mM 1-butanol, 0.13–10.25 mM 2-butanol, 2.56–9.71 mM isobutanol, 8.62–42.77 mM isoamyl alcohol in the Hungarian samples and 0.0–237.0 mM methanol, 0.0–17.0 mM 1-propanol, 0.0–1.2 mM 1-butanol, 0.0–0.2 mM 2-butanol, 0.0–23.6 mM isobutanol, 0.0–53.0 mM isoamyl alcohol in the Polish samples4,46. Assuming that spirits that also contained 40% ethanol had concentrations of AAs ranging up to the maximal concentrations of AAs reported in these spirits, we calculated how much would be needed to reach minimal inhibitory concentrations of AAs in the blood via Widmark’s equation: A ¼ C  p  r; where A is the amount of alcohol consumed (g), C is the blood alcohol concentration (g/L), p is the body weight (kg), r is Widmark’s factor (0.7 l/kg for men; 0.6 l/kg for women)50. Applying Widmark’s factor for men to these levels of AAs and a

product containing 40% ethanol, Table 1 presents the amount of recorded and unrecorded spirits that would have to be consumed to reach the minimal inhibitory concentrations of AAs (0.005 mM) in the blood and the corresponding blood ethanol levels. Considering those spirits samples that contained AAs, the volume of spirits needing to be consumed and the corresponding blood ethanol concentrations vary widely, according to the type of AAs and spirits concerned (Table 1). Where the volumes to be consumed are greater than 500.0 ml the estimate is not of practical biological relevance51. Table 1 also shows that the values at the lowest end of blood ethanol concentration range were usually more than 2.0 mM. At this and higher concentrations, the effect of ethanol can be expected to be predominant. However, values at the lowest range of blood ethanol concentrations were estimated to be lower (0.3 mM, 0.22 mM, and 0.7 mM) after consumption of

Aliphatic alcohols inhibit phagocytosis by monocytes 0.001–0.003 0.001–0.01 0.0–0.0002 0.0–0.0001 0.003–0.008 0.004–0.006 Methanol 1-Propanol 1-Butanol 2-Butanol Isobutanol Isoamyl alcohol

Both are described in the Discussion. AAs, aliphatic alcohols, n.d., no data. The volume of recorded and unrecorded alcoholic beverages and concentrations of AAs were estimated by Widmark’s equation. a The volume required to be consumed to reach 10 mM blood ethanol concentration was calculated to be 71.3 ml of distilled spirit containing 40% ethanol. b The concentration of AAs in distilled spirits is taken from previous research, see Ref.46. c The concentration of AAs in distilled spirits is taken from previous research, see Ref.47. d The concentration of AAs in distilled spirits is taken from previous research, see Ref.44.

0.66–2.09 0.85–6.59 0.0–0.11 0.0–0.05 2.29–5.53 2.44–3.99 0.051–0.116 0.005–0.107 0.0001–0.0003 0.0001–0.01 0.002–0.01 0.01–0.03 37–80 3.70–74 0.07–0.19 0.04–6.40 1.51–5.55 4.45–20 0.001–0.148 0.002–0.010 0.0–0.001 n.d. 0.010–0.040 0.020–0.055 0.6–102 1.1–8.3 0.0–0.4 n.d. 5.6–24 17–38 0.0–237 0.0–17 0.0–1.2 0.0–0.2 0.0–24 0.0–53

Aliphatic alcohol

0.0–0.343 0.0–0.025 0.0–0.002 0.0–4.0x106 0.0–0.034 0.0–0.077

Concentration of AAs in the blood (mM) Concentration of AAs in spirits (mM) Concentration of AAs in the blood (mM) Concentration of AAs in spirits (mM) Concentration of AAs in the blood (mM) Concentration of AAs in spirits (mM) Concentration of AAs in the blood (mM)

Samples collected in Russia (n ¼ 7, samogon) Samples collected in Poland (n ¼ 33, homemade spirits)

Concentration of AAs in spirits (mM)

Samples collected in Germanyd (n ¼ 27, Scotch whiskey) Samples collected in Germanyd (n ¼ 56, Kirschwasser)

Recorded alcoholic beverages

c b

Unrecorded alcoholic beverages

Table 2. Concentrations of aliphatic alcohols in distilled spirits and related blood levels after drinking sufficient volumea of distilled spirits to reach 10 mM blood ethanol concentration.

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unrecorded spirits containing high concentrations of methanol and 1-propanol (unrecorded Hungarian and Polish spirits, recorded German spirits, Table 1). In addition, blood ethanol levels following ingestion of unrecorded Hungarian and Polish spirits containing high amounts of isoamyl alcohol were also at the lowest end of concentration range, 1.0 mM and 1.2 mM, respectively (Table 1). Therefore, in these cases, methanol, 1-propanol, and isoamyl alcohol may affect phagocytosis. On the other hand, in the presence of 10.0 mM ethanol, the mixture of AAs significantly decreased phagocytosis at a concentration of 0.05 mM (Figure 6). To reach a blood ethanol level of 10.0 mM, consumption of 71.3 ml of spirit containing 40% ethanol is required. Ingestion of this volume would give rise to the blood concentrations of AAs presented in Table 2. As shown, the blood levels of methanol would vary among 0–0.051–0.116 mM, 0– 0.343 mM, and 0.001–0.148 mM following consumption of recorded and unrecorded spirits (Table 2). Similarly, the estimated blood concentrations of 1-propanol and isoamyl alcohol would be in the range of 0.005–0.107 mM, 0– 0.077 mM, and 0.02–0.055 mM in recorded and unrecorded spirits (Table 2). These AAs, when mixed, inhibited phagocytosis at a concentration of 0.05 mM, which was within the above-mentioned ranges. Therefore, at least methanol, 1propanol and isoamyl alcohol may inhibit phagocytosis in drinkers consuming recorded and unrecorded spirits that contain these AAs in sufficient concentration. This was estimated to be 34.3 mM or 110 g/hl methanol, 200 g/hl 1propanol, and 303 g/hl isoamyl alcohol. It is important to note that such an amount of methanol was measured in 3 of 33 Polish and 3 of 7 Russian spirit samples and isoamyl alcohol in 4 of 33 and 3 of 7 Russian spirit samples46,47. The next question was whether anyone would actually drink these quantities, ingesting sufficient quantity of AAs to inhibit phagocytosis by monocytes in vivo. Spirits produced from fruits and grains are widely available in the CEE countries, going under different names include ‘‘pa´linka’’ (Hungary), ‘‘sligovica’’ (Slovakia), and ‘‘samogon’’ (Russia, Ukraine, and Belorussia), and ‘‘tuica’’ (Republic of Moldova and Romania)2,4. Someone meeting the WHO criteria for an episodic heavy drinker would consume at least 60 g or more of pure alcohol on at least one occasion in a 7-d period1. This would involve consumption of 190 ml of spirit containing 40% ethanol. This easily exceeds the quantities required to inhibit phagocytosis for all AAs studied (except isobutanol). A large epidemiological study demonstrated that 420% of men in CEE countries are episodic heavy drinkers and many drink4200 ml spirits at least once every 2–3 weeks51. As these calculations and epidemiological studies show, it is reasonable to assume that alcohol abusers and episodic heavy drinkers (especially in CEE countries) will consume spirits in quantities whereby the concentrations of ingested AAs in their blood would reach levels sufficient to decrease phagocytosis by monocytes in vivo. By this, consumption of AAs in spirits may contribute to increased susceptibility to infectious diseases in these subjects. Clearly, further research is required to explore this question fully. On the other hand, it should be noted that some toxicokinetic studies have reported concentrations of higher alcohols and ethanol in the blood of alcoholics after ingestion of beverages containing aliphatic

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alcohols within the range of 0.01–0.10 mM and 10 mM, respectively52,53. However, the peak concentrations of methanol and 1-propanol in the blood of alcoholics have been reported to be as high as 10.0 mM and 0.22 mM, respectively52,53,54. Moreover, all the eight alcohols tested showed inhibitory effects at a concentration as low as 0.005 mM, which was below the range observed in alcohol abusers. In addition, they can increase the inhibitory effect of ethanol on phagocytic activity when combined with it. In summary, the results of the present studies indicate that AAs in spirits can inhibit phagocytosis by human monocytes in a concentration-dependent manner. Thus, it is possible that, alone or in combination with ethanol, these substances may inhibit phagocytosis at concentrations observed in alcoholics and episodic heavy drinkers thereby contribute to their increased susceptibility to infectious diseases. However, further research is needed to address this question.

Acknowledgements The authors thank Mrs. Mariann Kova´cs for the excellent technical assistance and Association of Hungarian PhD students for permission to present Figures 2–6.

Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the National Development ´ MOP 4.2.2./B-10/1-2010-0024, Agency (Contract no. TA ´ MOP 4.2.1./B-09/1/KONV-2010-0007), TA ´ MOP-4.2.2.ATA 11/1/KONV-2012-0031, and Ministry of National Resources (Contract no. 1EVJ 1NB0 EGPL 320). The project was cofinanced by the European Union and the European Social Fund.

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Aliphatic alcohols in spirits inhibit phagocytosis by human monocytes.

A large volume of alcoholic beverages containing aliphatic alcohols is consumed worldwide. Previous studies have confirmed the presence of ethanol-ind...
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