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DOI: 10.1111/jdv.

13620 JEADV

ORIGINAL ARTICLE

Precipitation of free fatty acids generated by Malassezia – a


possible explanation for the positive effects of lithium
succinate in seborrhoeic dermatitis
P. Mayser,1,* S. Schulz2
1
Center for Dermatology, Venerology and Allergology, Justus Liebig University, Giessen, Germany
2
Institute of Inorganic and Analytical Chemistry, Justus Liebig University, Giessen, Germany
*Correspondence: P.A. Mayser. E-mail: peter.mayser@derma.med.uni-giessen.de

Abstract
Background Lithium succinate and gluconate are effective alternative options licensed for the topical treatment of
seborrhoeic dermatitis (SD).
Objective Their mode of action is not fully elucidated. Minimal inhibitory concentrations against Malassezia (M.) yeasts,
which play an important role in SD, are very high.
Methods An assay based on the hydrolysis of ethyl octanoate enables us to test the hydrolytic activity of reference
strains of the species M. globosa, M. sympodialis and M. furfur solely without interference by fungal growth as the free
octanoic acid generated has antifungal activity.
Results In this assay the presence of alkali salts (lithium, sodium and potassium succinate resp.) in concentrations of
2%, 4% and 8% does not influence hydrolytic activity but the availability of the generated free fatty acid in a dose-
dependent manner which was analysed by means of high-performance thin layer chromatography and densitometry.
This was best effected with the lithium, followed by the sodium and only to a low degree by the potassium salt. As shown
by attenuated total reflection Fourier transform infrared spectroscopy the free fatty acid reacted to the respective alkali
soap and precipitate from solution. The alkali soaps could not be utilized by the M. spp. as shown in a modified Tween
auxanogram and in lack of fungal growth by ethyl oleate in the presence of 8% lithium succinate.
Conclusion The effect of lithium succinate on growth of M. yeasts and presumably in SD can be explained by a precip-
itation of free fatty acids as alkali soaps limiting their availability for the growth of these lipid-dependent yeasts.
Received: 4 December 2015; Accepted: 13 January 2016

Conflicts of Interest
The authors state no conflict of interest.

Funding Sources
None declared.

Introduction daily lithium succinate ointment was associated with signifi-


Seborrhoeic dermatitis (SD) is a chronic relapsing inflammatory cantly greater reductions in erythema, scaling and the percentage
skin disease especially in areas rich in sebaceous glands.1 Until of the area of the skin involved.6 In a small, randomized trial
now aetiology and pathophysiology of SD are not entirely eluci- involving 12 patients, lithium succinate was significantly more
dated. Correlation of SD flares with proliferation of Malassezia effective than placebo for the treatment of lesions in HIV-posi-
(M.) species and clinical response of SD to antifungal agents are tive patients.7 In 129 patients with facial lesions, application
indicators that M. yeasts play an aetiologically important role.2,3 twice-daily of lithium gluconate was shown to be more effective
Therefore most compounds used today in SD’s therapy have an than placebo in an 8-week trial, and it was shown to be superior
antifungal mechanism of action.2 Topical lithium succinate and to 2% ketoconazole in an 8-week non-inferiority trial involving
lithium gluconate are effective alternative agents for the treat- 288 patients with facial lesions; in the latter study, complete-
ment of SD in areas other than the scalp.4–9 In a crossover, pla- remission rates were 52% with the use of lithium gluconate and
cebo-controlled trial of lithium succinate involving two 4-week 30% with the use of ketoconazole, but ketoconazole was applied
treatment periods separated by a 2-week washout period, twice- only twice weekly.8,9 However, the way lithium salts work is

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2 Mayser and Schulz

poorly understood. In comparison to antifungals the minimal late, CAS 106-32-1; Sigma–Aldrich, St. Louis, MO, USA). By
inhibitory concentrations (MIC’s) against M. spp. are very high. determining cell counts with a Neubauer chamber (dilution
Boyle et al.4 reported that there was no growth inhibition of M. media xylene) suspensions were prepared to give a density of
furfur in vitro until a concentration of 150 mg lithium succinate 105 cells/lL respectively.14
per litre (~1.21 mmol/L; MW 124.02 g/mol) was reached. Leem- SA was poured out on small Petri dishes (3 cm in diameter)
ing and Burton10 found MIC’s between 3 and 100 mmol/L, and overlaid with 50 lL of the inoculated ethyl caprylate. In
which was confirmed in the study by Nenoff et al.11 who addition, SA was prepared with the following alkali salts in a
reported MIC’s between 1250 and 10 000 mg/L – 0.125% to 1% concentration of 2%, 4% and 8% (w/v), respectively, and pro-
lithium succinate.. These are much higher concentrations than cessed as well (Table 2). After incubation at 32 °C for 0, 1, 3, 6,
those which are normally used for susceptibility testing. How- 24, 48 and 72 h, respectively, the suspension was taken up with
ever, when compared to the 8% lithium succinate ointment in 1 mL chloroform (CHCl3) and transferred to gas-dense tubes.
the commercially available preparations, the investigated con-
centrations are even far below this value. Thin-layer chromatography and densitometry
Free medium and short chain fatty acids are known to have High-performance thin-layer chromatography (HPTLC) was
broad antimicrobial activity. Most recently a nearly identical performed to determine the amount of remaining ethyl caprylate
antimicrobial effect was found with the ethyl ester derivatives of and free octanoic acid generated by the different Malassezia spp.
these fatty acids, but only against Malassezia (M.) spp., not at different time points.
against Candida spp.12 Obviously these esters are hydrolysed by 2 lL of each sample taken up with CHCl3 were applied to
M. enzymes, thus generating a selective activation of antimicro- HPTLC 60-Plates (Merck) by means of a Camag Linomat IV
bial activity. Octanoic acid ethyl ester (CAS 106-32-1) was found (Camag, Muttenz, Switzerland) and fractionated by HPTLC (elu-
to be most effective. Consequently, an assay based on the hydrol- tion media hexane : diethylether : formic acid 80 : 20 : 2 v/v/v).14
ysis of ethyl octanoate enables us to test the hydrolytic activity of After development, each plate was dipped in a solution of
reference strains of the species M. globosa, M. sympodialis and primuline (Sigma) (Stock 0.1% in methyl alcohol; final solution
M. furfur solely without interference by fungal growth as the free in acetone 1 : 10 v/v) according to Wright.15 Results were quan-
octanoic acid generated has antifungal activity. Surprisingly the tified densitometrically with a Camag TLC Scanner 3 using
exo hydrolases of these species were active more than 72 h. Our Camag Software (Camag) and parallel processing of authentical
first hypothesis was that Lithium might influence hydrolase ethyl caprylate and octanoic acid standards (Sigma). Absolute
activity of the Malassezia yeasts. However, the data achieved values were obtained by comparison with corresponding stan-
with our new assay suggest that growth inhibition of M. yeasts dard curves. Amounts of FFA present at the time t = 0 (primar-
by lithium salts might not be based on direct antifungal effects ily oleic acid due to the inocula) were subtracted from each
or inhibition of hydrolytic activity but on a reduction in the measured value.
availability of free fatty acids (FFA), which are essential for the
growth of lipid-dependent M. yeasts. Influence on growth
Instead of octanoic acid ethyl ester oleic acid ethyl ester (ethyl
Materials and methods oleate, CAS 111-62-6, Sigma–Aldrich) was used for culturing as
Reference strains of the following Malassezia species were free oleic acid can be utilized by M. spp. M. sympodialis CBS
included in the investigation (Table 1). They were maintained 7222 was inoculated in a density of 105 cells/lL and incubated
on modified (m) Dixon-agar.13 as described above with or without 8% lithium succinate in the
SA. Cells were counted after 24, 48 and 72 h.
Culturing
Cultures were first grown aerobically on selective agar for patho- Analysis of smear
genic fungi (SA, Merck, Darmstadt, Germany), overlaid with a Presence of alkali salts of succinic acid during incubation
thin layer of olive oil at 32 °C. After 8–10 days cells were har- induced formation of a smear starting after 3–6 h of incubation.
vested and suspended in octanoic acid ethyl ester (ethyl capry- The smear was washed off the cultures using chloroform and
Table 2 Alkali salts tested
Table 1 Strains tested
Alkali salt CAS Obtained from
Species Strain tested Lithium succinate 16090-09-8 generous gift of Dr. Furrer,
M. furfur CBS* 7019 Louis Widmer SA, Schlieren, CH
M. sympodialis CBS* 7222 Sodium succinate 150-90-3 Sigma–Aldrich, St. Louis, USA
M. globosa CBS* 7966 Potassium succinate 22445-04-1 MP Biomedicals, Santa Ana, USA
Lithium chloride p.A. 7447-41-8 J.T. Baker, Deventer, NL
*CBS – Centraalbureau voor Schimmelcultures (www.cbs.knaw.nl).

JEADV 2016 © 2016 European Academy of Dermatology and Venereology


Precipitation of fatty acids by lithium salts 3

was analysed by means of attenuated total reflection Fourier (a)


50
transform infrared spectroscopy (ATR-FTIR spectroscopy).16 45
In brief a small amount of smear and standards, respectively,

Ethyl octanoate [µl]


40
were given on the ATR diamond of the ATR-FTIR (Bruker Alpha 35
30
equipped with ATR Platium Diamond, a MIR Globar, KBr beam 25 Control
splitter, RT-DLaTGS detector). IR spectra were collected from 20
Li 2%
15
400 to 4000 cm 1 with 2 cm 1 resolution. 50 scans were 10 Li 4%

summed, resulting IR spectra display absorbance of the analyte. 5 Li 8%


0
0 1 3 6 24 48 72
Generation of the alkali soaps for feeding experiments Time (h)
(b)
Alkali soaps were generated from oleic acid (Aldrich, CAS 112- 35 Control
80-1) by an equimolar acid-base reaction with potassium

Free octanoic acid [µl]


30 Li 2%

hydroxide (Sigma Aldrich, 99%, CAS 1310-58-3), sodium 25 Li 4%


Li 8%
hydroxide (Carl Roth, 99.99%, CAS 1310-73-2) and 20
lithium hydroxide monohydrate (CAS 1310-66-3) respectively. 15
0.005 mol of oleic acid were solved in 5 mL methanol (Merck, 10
Uvasol quality, CAS 67-56-1) and 0.005 mol of base solved in 5
5 mL water (LC quality, Fluka, CAS 7732-18-5) were added to 0
0 1 3 6 24 48 72
the solution. Generated alkali oleates were dried for 2 h at 35 °C Time (h)
using a centrifugal evaporator (SavantTM SPD111 SpeedVac).
Among the different alkali soaps lithium oleate precipitated
Figure 1 (a) Hydrolysis of octanoic acid ethyl ester by CBS 7222
most rapidly from the organic-aqueous solution, followed by without or with 2,4 and 8% lithium succinate resp. x of two inde-
sodium oleate. Potassium oleate showed the highest solubility in pendent values (b) Generation of octanoic acid by CBS 7222 with-
this mixture. Oleic acid was used, as in case of hydrolysis the free out or with 2, 4 and 8% lithium succinate resp.; 
x of two
acid can be utilized by M. spp.17 Lithium, potassium and sodium independent values [results corresponding to hydrolysis of ethyl
octanoate in (a)].
oleate were used in a modified Tween auxanogramm according
to Gueho et al.13 In brief two loops of each 4- to 5-day-old M.
cultures were suspended in 2.0 mL of sterile demineralized nounced with lithium succinate, followed by sodium succinate,
water. This inoculum was added to 17 mL of a molten SA main- whereas FFA concentration was only minimal affect by potas-
tained at 50 °C, and the mixture was poured immediately in a sium succinate (Fig. 2a–c). Interestingly this correlates well with
9 cm Petri dish. After complete solidification, wells were made the experimentally observed solubility of these substances in
with a 3 mm diameter punch, four devoted to test the growth organic-aqueous mixtures. In contrast to lithium succinate with
using Tweens 20, 40, 60 and 80 (Sigma) clockwise around, with lithium chloride the effect was totally absent indicating an influ-
a mark to indicate the position of Tween 20, and a fifth hole in ence of the anion as well. Hydrolysis of the ester as well as gener-
the centre to test growth with the respective alkali soap dissolved ation of free octanoic acid was not influenced by any of the
in aq. dest. 1 : 10 (v/v). The wells were filled with approximately concentrations of lithium chloride tested (2%, 4%, 8% w/v).
10 lL of each product and incubated for 9 days at 32 °C. Presumably the organic anion of lithium succinate increases the
solubility of the salt in the ester making it more available for the
Results reaction with the free fatty acid.
Hydrolysis of ethyl octanoate by all Malassezia spp. tested led to
the generation of free octanoic acid in a time-dependent man- Influence on growth
ner. Hydrolysis started immediately and reached nearly 100% With ethyloleate as substrate growth could be observed in our
after 72 h (Fig. 1a). However, addition of the alkali succinate’s assay, while with the addition of 8% lithium succinate to the
resulted in a dose-dependent reduction in the amount of free medium growth was totally absent.
octanoic acid (Fig. 1b). As published recently12 free octanoic The feeding experiments with the modified Tween aux-
acid does not induce growth in the M. spp. tested. As hydrolysis anogramm showed that in contrast to the Tween’s the alkali ole-
of the ester could be visualized at least over the next 72 h, this ates could not be utilized by any of the Malassezia spp. tested.
assay allows measurement of the activity of malassezia exo
hydrolases without the amount of generated FFA influenced by Analysis of smear
yeast growth and fatty acid assimilation. The reduction in the amount of FFA was not due to a reduced
Free fatty acid concentration was also found to be dependent hydrolytic activity, but due to the formation of alkali soaps, pre-
on the alkali salt’s cation. Reduction in FFA was most pro- sumably through an acid-base reaction of octanoic acid with

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4 Mayser and Schulz

(a) Control
(b) Control
(c) Control
2% Li 2% Na 2% K
4% Li 4% Na 4% K
8% Li 8% Na 8% K
120 120 120
Relative abundance of

Relative abundance of

Relative abundance of
100 100 100
free fatty acid [%]

free fatty acid [%]

free fatty acid [%]


80 80 80
60 60 60
40 40 40
20 20 20
0 0 0
7222 7019 7966 7222 7019 7966 7222 7019 7966
Malassezia strain [CBS #] Malassezia strain [CBS #] Malassezia strain [CBS #]

Figure 2 (a–c) Amount of octanoic acid generated from ethyl octanoate by the different M. spp. The amount generated without the alkali
salt was set to 100% (control). (x of two independent values; difference< 5%). (a) Results obtained with lithium succinate. (b) Results
obtained with sodium succinate. (c) Results obtained with potassium succinate.

succinate. The presence of alkali soaps was confirmed by analysis spp. without the amount of generated FFA influenced by yeast
of the smear with ATR-FTIR spectroscopy (Fig. 3a–d). growth and fatty acid assimilation. The enzymes were still vital
for at least 72 h. We included reference strains of M. spp. (M.
Discussion globosa, M. sympodialis and M. furfur) which are commonly iso-
The exact aetiology and pathophysiology of SD are not yet clear. lated from lesions of SE.2,19 All strains showed hydrolytic activity
A complex scenario involving sebum production, lipid composi- of ethyl octanoate which started immediately. The presence of
tion on the skin surface, Malassezia (formerly Pityrosporum) alkali salts (lithium, sodium and potassium succinate resp.) in a
species and patient predisposition to immune or inflammatory concentration of 2%, 4% and 8% did not influence hydrolysis of
reactions is discussed.1,2 Clinically it has been shown that there the ester but the availability of the generated free fatty acid
is a decrease in Malassezia spp. population with antifungal treat- which reacted to the respective alkali soaps in a dose-dependent
ment in parallel with improvement in SE clinical signs.1–3 The manner. The highest reduction in FFA amount was found for
effectiveness of topical lithium succinate ointment in SD was the lithium salt, followed by the sodium salt. The potassium salt
shown in preliminary studies.4–9 However, the way of action is reduced the FFA amount only to a low degree. In addition to the
not entirely clear. In vitro growth of Malassezia spp. was inhib- cation the anion seems also of importance. In contrast to lithium
ited by lithium salts but in concentrations much higher than succinate no effect could be seen with lithium chloride. It is
those which are normally used for susceptibility testing.4,10,11 assumed that the addition of alkali succinate results in the
Therefore, it was suggested that lithium succinate presumably deprotonation of the FFA and the formation of the alkali soap
acts as an anti-inflammatory agent blocking the release of (Fig. 4). As experimental confirmed these soaps exhibit a quite
arachidonic acid.18 In this article we describe a new in vitro test different solubility. Precipitation of the soaps from solution is
system which allows the measurement of exo hydrolases of M. expected to shift the chemical equilibrium of the protonation

(a) (c)

Figure 3 (a) ATR-FTIR spectrum of a)


smear collected from culture incubated on a
(b) (d)
medium with 8% lithium succinate (treated
culture) (b) Lithium octanoate standard (c)
smear collected from non-treated culture (d)
octanoic acid standard. Interpretation: (a)
and (b) as well as (c) and (d) are very similar.
Hence, lithium octanoate is formed from
octanoic acid in the treated culture, but not
in the non-treated culture.

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Precipitation of fatty acids by lithium salts 5

(a) Hydrolase
our results have shown that exo hydrolases may be still active
O OH
H3C CH3 H 3C + H 3C OH for a certain time without vital fungus.
CH3
O O
2 Direct inhibition of Malassezia exo lipases and hydrolases,
(b) O O which concept is to our best knowledge not realized today.
H3C OH + HO O- Li+ OH + O
OH -
However, our new test system could help in screening appro-
O O O O Li+
priate compounds.
Figure 4 (a) Enzymatic hydrolysis of ethyl octanoate to ethanol 3 Reduced availability of FFA generated by Malassezia lipases
and octanoic acid; (b) formation of lithium octanoate from octanoic and hydrolases. As these are essential compounds for the
acid and lithium succinate (simplest reaction is displayed). obligatory lipid-dependent yeasts it results an indirect anti-
fungal effect. Furthermore, other toxic products of lipase
reaction in favour of the octanoate, hence removing more FFA activity may be neutralized in such a reaction. Our data have
from solution the lower the solubility of the respective alkali shown that the effectiveness of lithium succinate, which is
soap. For lithium succinate the most effective dosis was 8% cor- used as effective treatment in the topical therapy of SD for
responding to that, which was already shown to be effective in more than 20 years, is based at least in part on this concept.
clinical studies. The alkali soaps could not be utilized by the M. Alkali succinate reacts with the FFA to generate alkali soaps
spp. as shown in a modified Tween auxanogram and in lack of which cannot be utilized by M. spp. anymore. This reaction
fungal growth by ethyl oleate in the presence of 8% lithium suc- is further favoured by precipitation of the alkali soaps. This is
cinate. Furthermore, fatty acids gain transformed to alkali salts best effected with lithium succinate, since the corresponding
water solubility and can be rinsed off. Therefore, the effect of lithium soaps feature the lower solubility. Furthermore, fatty
lithium salts on growth of M. yeasts and presumably in SD can acids as transformed into alkali soaps gain water solubility
be explained by a limited availability of FFA which are essential and can be rinsed off which results in a further effective
for growth of these lipid-dependent yeasts. With the exception reduction in the pathogenic important products of lipase
of M. pachydermatis all 14 Malassezia species known so far activity in SD.
require an external lipid source for growth, i.e. they are obligato-
rily lipid-dependent.20 Lipid dependence is known to be based Acknowledgements
on a defect in the synthesis of myristic acid (C14:0), which serves Anne Gries is gratefully acknowledged for excellent technical
as the precursor of long-chained fatty acids. At the molecular assistance.
level, this metabolic deficiency can be explained by the absence
of a gene encoding fatty acid synthase in the genomes of M. glo- References
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