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Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 1312 Clin Biochem Rev 38 (1) 2017
Review Article
Working Up a Good Sweat – The Challenges of Standardising Sweat
Collection for Metabolomics Analysis
*Joy N Hussain,1 Nitin Mantri,2 Marc M Cohen1
1School of Health and Biomedical Sciences, RMIT University, Bundoora, Vic. 3083; 2Health Innovations Research Institute,
School of Applied Sciences, RMIT University, Bundoora, Vic. 3083, Australia.
*For correspondence: Dr Joy Hussain, joyhussain9@gmail.com
Abstract
Introduction
Human sweat is a complex biouid of interest to diverse scientic elds. Metabolomics analysis of sweat promises to improve
screening, diagnosis and self-monitoring of numerous conditions through new applications and greater personalisation of medical
interventions. Before these applications can be fully developed, existing methods for the collection, handling, processing and
storage of human sweat need to be revised. This review presents a cross-disciplinary overview of the origins, composition,
physical characteristics and functional roles of human sweat, and explores the factors involved in standardising sweat collection
for metabolomics analysis.
Methods
A literature review of human sweat analysis over the past 10 years (2006–2016) was performed to identify studies with metabolomics
or similarly applicable ‘omics’ analysis. These studies were reviewed with attention to sweat induction and sampling techniques,
timing of sweat collection, sweat storage conditions, laboratory derivation, processing and analytical platforms.
Results
Comparative analysis of 20 studies revealed numerous factors that can signicantly impact the validity, reliability and
reproducibility of sweat analysis including: anatomical site of sweat sampling, skin integrity and preparation; temperature
and humidity at the sweat collection sites; timing and nature of sweat collection; metabolic quenching; transport and storage;
qualitative and quantitative measurements of the skin microbiota at sweat collection sites; and individual variables such as diet,
emotional state, metabolic conditions, pharmaceutical, recreational drug and supplement use.
Conclusion
Further development of standard operating protocols for human sweat collection can open the way for sweat metabolomics to
signicantly add to our understanding of human physiology in health and disease.
Introduction
Human sweat is a biological uid (biouid) that is generating
increasing interest across a diverse set of elds including
dermatology, paediatrics, toxicology, analytical chemistry,
forensic pathology, psychiatry, illicit drug testing and infectious
diseases. Currently sweat is primarily used in clinical medicine
for chloride sweat testing which is used in the diagnosis
of cystic brosis (CF). Additionally, some centres around
the world use a sweat patch for monitoring drugs of abuse,
while others have developed an indicator test (Neuropad) to
detect peripheral neuropathy in the foot sweat of diabetics.1-3
Aside from these applications, the use of sweat in medical
practice is limited in part due to challenges involved with
sweat collection and the range and reproducibility of testing.
This is likely to change as advances in analytical technology
methods within metabolomics and other related ‘omics elds
allow more complex physiological information to be derived
from smaller amounts of sweat with less arduous processing.
This is leading to a greater understanding of the physiology of
human sweating and the skin’s excretory pathways in relation
to metabolites, pathogens, and xenobiotics.4 Incorporation
of Bluetooth capabilities with some of the newer wearable
sweat electrolyte and metabolite detecting systems reects
even wider trends in applications to enhance personalised
analysis.5-7
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 1514 Clin Biochem Rev 38 (1) 2017
Each type of human biouid or tissue sample has its own
signature metabolome, but most of what is known about the
human metabolome is based upon ndings in the ‘serum/blood
metabolome’ and the ‘urine metabolome’. Further study and
standardised procedures are now required to characterise the
‘sweat metabolome’ and how it ts into the bigger picture of
the human metabolome, and whether the case exists for wider
application of sweat metabolomic testing.
When applying a metabolomics approach to analysing human
sweat, a number of variables need to be examined within the
context of the origins, composition, physical characteristics
and functional roles of sweat. These variables include:
sweat induction and sampling techniques, timing of sweat
collections, sweat storage conditions, and laboratory aspects
such as metabolic quenching, extraction, concentration,
fractionation, separation and other processing methods
applicable to sweat. Exploring these variables within the
framework of newer laboratory analytical platforms that
optimise qualitative and quantitative detection of sweat
metabolites will pave the way forward to make more rigorous
and meaningful comparisons of sweat metabolomics studies.8
Standardising the collection, handling, processing and storage
of sweat for further metabolomics analysis is vital to this
endeavour and working out the further steps necessary to
achieve this standardisation is the focus of this review.
Background – Metabolomics
Metabolomics is the multidisciplinary science involving
the measurement and analysis of low molecular weight
metabolites such as electrolytes, sugars, lipids and other
compounds that exist in a selected biouid, cell, tissue or
organism under a given set of physiological conditions. It has
its roots in the works of many biochemists who pioneered
the discovery and detection of various vitamins in the 1940s
and progressed the concepts of ‘metabolic variance’ and
‘biochemical individuality’.9-12
The exact number of unique metabolites in the human
metabolome has yet to be rmly established, but it is
generally thought that there is a lower number of metabolites
in the human metabolome (>3,500) compared with the total
number of genes (>30,000 in genome), RNAs/transcription
factors (>30,000 in transcriptome) and proteins (>100,000 in
proteome).13 Small changes in the transcriptome may translate
into more amplied changes in metabolites.14 With presumed
fewer total metabolites to analyse and a potentially more
amplied signal to be detected, the power and potential of
metabolomics to pick up minute but signicant health-related
changes holds promise.
As with all newly emerging elds, within metabolomics
there is multiplicity and various expansions of terminology.
Although metabolomics and metabonomics are often used
interchangeably in the literature, metabonomics technically
refers to the study of the interactions of metabolites over
a timeframe in a complex system.15 Fluxomics refers to an
extension of metabolomics, in which metabolomics is applied
at various experimental time points generating kinetic data
which can then be used to study metabolic pathway uxes.16
Exposomics, another extension of metabolomics, refers to
identifying metabolites linked to environmental risk factors
for disease.17 Metabolites can be classied into two categories:
endogenous metabolites (synthesised and utilised within a
biological system) and exogenous metabolites (imported
from outside the biological system into the cell, such as drugs,
xenobiotics and nutrients).13,16 The Human Metabolome
Project (HMP) led by Dr David Wishart of the University
of Alberta in Canada published a rst draft of the human
metabolome in 2007 which consisted of 2180 metabolites,
1200 drugs and 3500 food components.18 A growing list of
ndings additional to the HMP is being compiled and veried
on the Human Metabolome Database – a freely accessible
and continually updated web resource (http://www.hmdb.
ca/).11,13,19 Not all known human metabolites can be found
in any given biouid because different biouids serve
different functions and play different metabolic roles. As of
November 2016, the HMP had identied and/or quantied
over 3848 metabolites: 440 metabolites in cerebrospinal uid,
1233 metabolites in saliva, 2287 metabolites in blood, 1746
metabolites in urine, 695 metabolites is faeces and over 172
metabolites in other tissues and biouids including sweat.19
The methodology of metabolomics can be divided into
different conceptual approaches such as targeted analysis,
global metabolite proling, metabolomics and metabolic
ngerprinting/ metabolic footprinting.20 A targeted
metabolomics approach involves a targeted search and
quantitative analysis of a set number of known metabolites or
substances that play a particular role, much like a typical clinical
laboratory test. Global metabolite proling is untargeted
and comprises an analysis of all measured metabolites or
substances, including those known and unknown, which
make up a metabolic prole of the total complement of
metabolites in a particular sample.13,20,21 Metabolomics
utilises complementary analytical methodologies such as
liquid chromatography-dual mass spectrometry (LC-MS/
MS), gas chromatography-mass spectrometry (GC-MS),
and nuclear magnetic resonance (NMR) spectroscopy
in a coordinated attempt at global metabolite proling.20
Metabolic ngerprinting refers to the metabolic ‘signature’ or
mass prole of the biouid or tissue sample of interest which
is then compared in a large sample population to screen for
differences between samples. When signals or signicant
differences can be detected, the metabolites are then identied
and the biological relevance of these particular metabolites
can be more easily elucidated. Metabolic footprinting is
analogous to metabolic ngerprinting except the differences
detected involve focus on extracellular metabolites.15
Sweat Origins, Components and Functions
Sweat Denitions
Whole body sweat is a complex mixture of cumulative
secretions from millions (1.6–5 million) of eccrine, apocrine,
apoeccrine and sebaceous glands as well as bacteria, yeast,
fungi, other microbiota and cellular debris that reside in
and on the largest organ of the human body – the skin.22
These microscopic glands dwell largely in the dermis and
hypodermis layers with secretory canals through which sweat
ows onto the skin surface and into hair follicles. Dening
sweat precisely is complicated by confusing nomenclature
across different disciplines in the scientic literature and
a lack of a biological systems-based approach to studying
sweat. Sweat collected from the skin surface in experimental
studies, especially older studies, is often referred to as
‘eccrine’ sweat because eccrine sweat glands are the most
numerous and ubiquitous glands in the skin, however many
sweat samples also contain potentially trace amounts of
apocrine, apoeccrine and sebaceous gland secretions (called
sebum), depending upon the body site of sampling. This
imprecision of nomenclature is the case in some toxicology
literature dealing with sweat patch testing of illicit drugs and
physiology literature studying electrolyte changes in exercise.
However, in the dermatology and cosmetic literature, ‘sebum’
can gure in addition to ‘sweat’ with more emphasis on the
underlying structures within the skin. The converse can also
be true with studies focused on collection of sebum. The
term ‘residual skin surface components’ (RSSC) is another
synonym of ‘sweat’ as it comprises potential sweat glandular
secretions and cellular debris (from stratum corneum –
outermost epidermal skin layer).23
Mindful of the semantics surrounding sweat, it is useful
to revisit the anatomy, histology and secretions of the four
known gland types that can contribute to sweat. This sets the
stage for better understanding and targeting of future studies
to fully characterise the sweat metabolome.
Eccrine Sweat Glands and Secretions
Eccrine sweat glands exist at birth and can be located all over
the body’s skin except on lips, on the nail bed and on some
elds of the genitalia (e.g. glans penis). They can average
100–200/cm2 body surface area, with higher densities (600–
700/cm2) on palms and soles, and at luminal diameters of
20–60 μm at skin openings.24,25 Eccrine glands consist of
single tubules ranging 4–8 mm in length that are generally
divided into 3 parts: (i) deep, coiled secretory portion in deep
dermis layers; (ii) upper dermal portion with straight and
coiled parts; and (iii) intra-epidermal part often referred to as
the acrosyringium. The dermal portion, or dermal duct, has
epithelial cells connected at numerous sites by desmosomes
and intercellular junctions that are believed to constitute a
barrier between the luminal and extracellular compartments.
The inner luminal cells contain various tonolaments while
the outer basal cells are surrounded by collagenous and
brocyte-rich sheathes.22,26
Eccrine sweat glands are classed as merocrine glands (Figure
1). Eccrine sweat gland secretions are released from cells as
an aqueous uid, without disintegration of cells, containing
various electrolytes, elements, ions, amino acids, proteins
and other known and unknown small molecules as outlined
in Figure 2.27,4 Composition varies with many factors: rate
of sweat production, transit time through the excretory
duct, aldosterone activity, physical training, psychological
states and acclimatisation to environmental temperatures.26
These give hints to underlying functions that have not yet
been fully determined. There also exists debate whether
secretory eccrine sweat is perhaps an isotonic ultraltrate of
plasma since sweat contains many of the same solutes found
in plasma, but at much lower concentrations.28 However,
based on a recent proteomics study of pooled sweat samples
collected from schizophrenic and control subjects, only 6
of 185 unique proteins identied in sweat were reported in
serum. The authors therefore argue that sweat is not merely
a plasma transudate and future metabolomics studies are
required to shed more light on this topic.29
Apocrine and Apoeccrine Sweat Glands and Secretions
Apocrine sweat glands also exist at birth but do not become
active until the androgenic stimulation of puberty.26 They are
conned to hairy body areas (i.e. axilla, mammary areola,
peri-umbilicus, perineal and genital areas) since they open and
secrete into adjacent hair ducts (e.g. apopilosebaceous ducts)
before secretions reach the skin surface. They are generally
larger than eccrine sweat glands with apocrine coil diameters
of ~800 µm compared to eccrine coil diameters of ~500
µm, both located in the dermis and hypodermis.22 Apocrine
ducts are relatively short and found in close proximity to hair
follicles. The density of apocrine glands is highly variable
with reports of 8–43/cm2 body surface area in one study of the
axilla.30 Two different types of cells are visualised in apocrine
glands: columnar secretory cells and myoepithelial cells. The
secretory cells are generally noted to be full of mitochondria
and different granules with convoluted cell membranes and
microvilli presenting towards the lumen.22
Apoeccrine sweat glands are a mixed type gland as the name
suggests and were rst described in 1987 by Sato et al.30 They
are also presumed to develop during puberty and be restricted
to hairy body areas. As many as 50% of all axillary sweat
glands are thought to be apoeccrine. Component cells of
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 1716 Clin Biochem Rev 38 (1) 2017
apoeccrine glands include eccrine secretory cells, apocrine
secretory cells and myoepithelial cells.30 Identication of
these morphologically distinct glands can be made with
specic protein markers (i.e. phalloidin,S-100, CD15).22
Apocrine and apoeccrine glands are both classed as apocrine
glands. With apocrine glands, secretion occurs via pinching-
off of the cell’s plasma membrane producing membrane-
bound vesicles, which helps to account for comparatively
more viscous secretions.22,26 Sato et al. determined that Na+
and K+ concentrations obtained from the isolated ducts of the
apoeccrine glands are curiously more similar to that of eccrine
sweat compared to apocrine sweat.30 While the composition of
apoeccrine sweat has not yet been fully elucidated, apocrine
sweat has been demonstrated to contain carrier proteins for
volatile odour molecules like volatile organic compounds
(VOCs) and pheromones with amino acid conjugates
produced by bacterial enzymes.22 Apocrine bromhidrosis
(more commonly known as BO or body odour) is thought
to be linked to large amounts (i.e. over 106 bacteria/cm2) of
resident microora such as aerobic cocci, diphtheroid species,
Corynebacterium species and Staphylococcus epidermidis.26,31
Sebaceous Glands and Sebum
Sebaceous glands are located in the skin of all surface areas
except for the palms of hands and soles of feet. They are
particularly numerous on the forehead, scalp, midline back,
chest, perineum and surrounding the orices of the human
body. Densities of up to 400–900 glands/ cm2 occur on the
face, especially in the T-zone area of the face which starts from
the midpoint and sides of the forehead, and extends downward
toward the middle of the nose, including the sides of the
nose and the midline part of chin.32 Sebaceous gland density
decreases towards the extremities of the body. Sebaceous
glands can be divided into two types: pilosebaceous glands,
when associated with hair follicles, and free sebaceous glands
seen mostly at transitional zones between skin and mucous
membranes. A well-known example of a free sebaceous
gland is the Meibomian gland of the eyelid. All sebaceous
glands consist of single or multiple lobules, or acini, with
ducts emptying into a main sebaceous duct. Secretory lobules
contain sebaceous gland cells, or sebocytes, that are excreted
in their entirety as part of the holocrine gland status. Maturing
sebocytes have been visualised to accumulate high lipid
content as they migrate from periphery to gland duct.33
Like apocrine sweat, sebum is thought to play a role in the
generation of pheromones and body odour in its interactions
with skin-residing bacteria and yeast of the microbiome.34
Sebum, however, is even more lipid-based, containing
triglycerides and fatty acids (together 57% of contents) as
well as cholesterol, wax esters, squalene, keratin, cellular
debris, anti-microbial lipids, antioxidants, coenzyme Q10,
vitamin E and other various metabolites of fat-producing
cells.34,35 Interestingly, the lipids in sebum would seem to
originate from both sebocytes and keratinocytes, with studies
identifying differences based upon cholesterol and squalene
conversion enzymes.34
Collected Sweat
Setting aside the above distinctions in gland origins, the
vast majority of sweat studies in the literature have analysed
a collective form of sweat with eccrine gland secretions
predominating. Depending upon location of sweat sampling
and various cleaning and collection strategies used during
sampling or processing, trace amounts of sebum and/or
apocrine and apoeccrine gland secretions, cellular matter from
the epidermis and associated ~1012 skin microbes, as well
as other metabolites like xenobiotics may feature in sweat
samples.36 The systems-based approach of analysing sweat
with metabolomics offers the prospect of uniting all these
different subcomponents of sweat. With such a metabolomics
approach, studies of ‘normal’ sweat obtained from ‘healthy’
people have detected highly variable metabolite compositions
with large numbers of different small molecules, of both
microbial and human origin, in a primarily water-based
(~99%), relatively acidic (mean pH 6.3) solution (Figure 2).37-40
This rich complexity of sweat content hints at its functions
both at the level of the skin and at the level of the organism
as a whole.
Functions of Sweat
Temperature and Fluid Homeostasis
Sweat is integral to the regulation of body core temperature
by water evaporative heat dissipation. Blood ow regulation
and vasodilation of supercial blood vessels largely
contribute to this homeothermic control and the nding that
eccrine sweat production is under the control of cholinergic
and, to a lesser extent, adrenergic innervation is consistent
with this hypothesis.41 Various stimuli of this system include
temperature, emotions, intellectual stimulation and gustatory
stimulation.42 Sweat volumes vary widely as a result. Global
insensible uid losses can be approximately 1000 ml daily
for the whole human body, including more than half of uid
losses through the skin via perspiration with the remaining
losses being through the lungs.4,40 However, there are reports
of individuals perspiring up to several litres per hour, 12 L per
day under certain extreme physiological conditions.4,35,43
Eccrine sweat activity appears intermittent over a large
portion of the body: cycles of periodic discharges alternating
with pauses occurring from <1 to 12 geyser-like emissions
per hour with single sweat gland emissions recorded every
3.3 min in one recent study.41 This activity differs among
(a) Merocrine
secretion -
Eccrine Sweat Glands
(b) Apoocrine
secretion -
Apocrine and
Apoeccrine
Sweat Glands
(c) Holocrine
secretion -
Sebaceous Glands
Secretion
Secretory vesicle
Golgi complex
Nucleus
Pinched o
plasma
membrane-bound
vesicle is secretion
Sebocyte dies
and becomes
secretory product
– sebum
Sweat Gland Secretion Patterns
Figure 1. Sweat gland secretion patterns.
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 1918 Clin Biochem Rev 38 (1) 2017
individuals, environmental circumstances and body sites
with approximately 50% of total body volume of sweat being
thought to be produced by the trunk, 25% by the legs and
25% by the head and upper extremities.1,48 Even in cases of
profuse sweating, it is thought that only approximately 50%
of sweat pores release sweat at any given time, except for the
palmoplantar regions where the sweat gland activity is largely
synchronised.44 How these ndings t within sweat’s overall
functions in the human body is still unclear.
In contrast to eccrine glands, apocrine gland activity is
reported to be more continuous in its uid secretions, while
still receiving predominantly cholinergic and some adrenergic
innervation.22,42 Apoeccrine gland stimulation by physiological
and pharmacological stimuli appears to be distinct from those
controlling eccrine or apocrine glands. Apoeccrine glands
respond quickly to psychological stress and are thought to
more signicantly contribute to the abundant sweat produced
in the axilla.25,30
Sweat Electrolyte Regulation
Another important component of sweat that impacts human
uid balances is sodium. The concentrations of Na+ in sweat
can be highly variable, ~20–100 mmol/L, and some individuals
can lose an estimated 4–6 g of Na+ per day, equivalent to 12–
15 g of NaCl daily through sweating, especially if working in
moderately hot conditions.43 Eccrine gland duct cells reabsorb
several ions, including Na+ and Cl-, via a number of known
anion exchangers such as Na+/K+-ATPases (on basolateral
membranes), cystic brosis transmembrane conductance
regulators (CFTRs, mutations of which provide the basis for
Cystic Fibrosis Sweat Chloride testing), carbonic anhydrases
II, and vacuolar proton pumps (V-H+-ATPase).22 Sweat Na+
and Cl- concentrations have been documented to increase with
age to 12–19 years then stabilise thereafter.45,46 Sweat Na+,
Cl-, and K+ concentrations also have reported body regional
variations.47
Acid-base homeostatic mechanisms are presumed to be
involved in sweating since sweat is more acidic than plasma,
with pH ranges of 4–6.8 reported in various studies.1,4 It is
noted that with increased ow rates following exercise or at
temperatures above 31 °C, sweat pH increases to upper limits
of approximately 6.8, which is still more acidic than plasma.47
Some non-ionised basic drugs diffuse into sweat and become
ionised as a result of the lower pH of sweat, although the
exact mechanisms have not been fully elucidated. This has
led to projections of these non-ionised basic drugs displaying
free-drug (or molecule) sweat-to-plasma (S/P) ratios of >1, as
in the case of ammonia with reported S/P ratios of 20–50.48,49
Skin Protection
Sweat also provides lubricating, water-proong, antimicrobial
and skin barrier-promoting properties that support skin in the
rst line of defence against many environmental insults. In
extreme hot conditions, the lipid-rich secretions of apocrine
and sebaceous glands can emulsify sweat produced by
eccrine glands to create a hydrolipid lm that is not as readily
evaporated. This is thought to be of importance in delaying
dehydration. In colder conditions, the lipid nature of sweat
becomes more solid and, in coating the hair and skin, sources
of unwanted moisture like rain or snow can theoretically
be more effectively repelled.50 Palmar hydration, which is
directly linked to eccrine sweat production, increases the skin
friction coefcient which therefore improves the adherence
of hands to objects and contributes to a heightened sense of
touch.41 Sweat also contains antimicrobial peptides (AMPs)
like dermicidin, lactoferrin, and LL-37, an AMP of the
cathelicidin family, which serve to control certain pathogenic
bacterial counts on the skin surface.4,41 However, the precise
qualitative and quantitative content of skin microbiota and
associated microbe-microbe and microbe-host dynamics via
sweat are areas of active research with early ndings hinting
at rich metabolic inter-relationships with impacts on skin
integrity, especially in skin inammatory states.36
The free amino acid composition of sweat is curiously
different from other biouids. Data from a recent study
suggest the amino acid content of sweat is remarkably similar
to the amino acid content of an epidermal protein, prolaggrin.
Since prolaggrin is thought to be the key contributor of free
amino acids making up the natural moisturising factor within
the stratum corneum, it is postulated that sweat plays a role
via interactions with prolaggrin in maintaining the barrier
integrity of human skin.51
Immune System
Sweat has links to many immune-mediated mechanisms.
Skin epithelial cells interact with various external stimuli to
produce cytokines, and sweat directly activates epidermal
keratinocytes to produce various cytokines using in vitro
models with cultured human keratinocytes from surgically
discarded neonatal skin samples.39 It is postulated that sweat
may play both benecial and pathological roles in immune-
mediated communications. For example, sweat is well-
recognised in exacerbating atopic dermatitis (AD) lesions
and is associated with increased itching (pruritus) which
has associations with enhanced expression of IL-31 (newer
member of IL-6 family of cytokines) in tissue samples of
exacerbated AD lesions.52 Sweat also contains cystatin A, a
proteinase inhibitor of bacterial cysteine proteases. Given
these exogenous proteases are known to break down the
epidermal barrier, cystatin A in sweat may serve both immune
Lipid/Hydrophobic Content < 1% - Primarily Sebum,
Apocrine Sweat origins:
lipids,
glycoproteins,
steroid hormones,
nitrogen,
lactate,
pheromones,
VOCs,
proteins/enzymes/cytokines,
triglycerides,
fatty acids,
antioxidants,
vitamins,
cholesterol,
cholesterol esterases,
wax esters,
squalene,
and more.
Aqueous/Hydrophilic Content > 99% -
Primarily Eccrine Sweat origins:
water,
sodium,
potassium,
chloride,
bicarbonate,
urea,
glucose,
magnesium,
lactate,
iron,
copper,
zinc,
calcium,
phosphate,
manganese,
chromium,
cobalt,
nickel,
iodine,
molybdenum,
amino acids,
vitamins,
BPA,
Phthalates,
heavy metals –
lead,
cadmium,
mercury,
arsenic,
foreign antigens,
and more.
Cellular Debris, Bacteria, Yeast, etc.
Metabolomic Sweat Content
Figure 2.
Metabolomic sweat content.
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 2120 Clin Biochem Rev 38 (1) 2017
and skin protective roles.53 Quantitative levels of IL-1α, IL-
1β, IL-6, TNF-α, IL-8 and TGF-β have been measured in
human sweat although the precise cellular origin of these
cytokines is still unknown and could be derived from sweat,
blood or epidermal cells.54
Excretion Functions and Drug Delivery Mechanisms
While the excretory function of sweat has previously been
considered negligible compared to the kidney, recent studies
challenge this notion. There is evidence that several toxic
elements and xenobiotics may be preferentially excreted
through human sweat.55-58 Some studies report arsenic,
cadmium, lead and mercury being excreted in appreciable
quantities via sweat, with the rates of excretion matching
or exceeding urinary excretion.58 Furthermore, while excess
dietary nicotinamide cannot be eliminated through urine
because of its reabsorption by the renal tubules, it can be
effectively excreted by sweat glands.35 Many pharmaceutical
drugs are also excreted via sweat and the role of sweat
patch technology in monitoring illicit drug use is based on
dozens of studies examining the pharmacodynamics and
pharmacokinetics of amphetamines, cocaine, cannabis,
opiates and associated metabolites excreted in sweat.1,48 Drug
binding to various skin fractions and reabsorption of drugs
from pooled sweat on skin has also been observed. The
relative concentration of unmetabolised drugs is reported to be
occasionally higher in sweat than in blood, urine or saliva.59,60
The above ndings suggest that molecules of drugs/
metabolites/xenobiotics may reach the skin surface from
blood by various proposed routes: via sweat or sebum by
active or passive inter- and/or transcellular mechanisms;
and transdermal migration through lipid bilayers of stratum
corneum.40 The second mechanism could be linked to the
concentration gradient in which only the free fraction of drug/
metabolite/xenobiotic, unbound to proteins, diffuses through
lipid membranes from plasma to sweat. Thus, it seems that
the physical nature (i.e. molecular mass, protein-binding,
pKa and lipophilicity) of each particular drug/metabolite/
xenobiotic plays a role in how much ends up in sweat. In
fact, dermatologists routinely take advantage of this scenario
when treating cutaneous fungal infections with oral antifungal
medications such as ketoconazole, terbinane or uconazole.
It is often recommended to exercise to induce sweating while
taking these oral antifungals since the drugs are transported
to the skin surface by eccrine sweat and/or sebum and then
often reabsorbed, thus optimising drug delivery to site of
infection.61-63
Metabolic and Infectious Diseases
The alteration of sweat with different pathological conditions
makes sweating a useful clinical indicator for various
conditions. Over-sweating (hyperhidrosis) and under-
sweating (hypohidrosis), whether regional or systemic, may
represent warning signs for systemic conditions or diseases.
Decreased sweat production involving the feet is the basis
for the Neuropad indicator test for diabetic peripheral
neuropathy.3 Impaired overall sweating is associated with pre-
eclampsia and thought to be related to decreased clearance of
plasma vasoactive amines.64 Female menopause is commonly
associated with ‘hot ushes’ linked to increased sweat
production.65
Night sweats can indicate serious systemic infections (e.g.
tuberculosis) and malignancy, while local hyperhidrosis
around a bite site can indicate toxic envenomations such as
occurs with Australian redback spider bites.66 Hypoglycaemia,
hyperthyroidism, hypercapnia and vagus nerve stimulation
can all lead to stimulation of eccrine sweat production and
alterations in local sweating may arise directly from certain
skin conditions.26 For example, some hyperkeratotic disorders
such as pityriasis versicolor and psoriasis interfere with
the excretion of sweat and are associated with decreased
sweat output as visualised with skin capacitance imaging of
lesions.44 Abnormalities in the transport of sweat onto the
skin’s surface may also cause a severe prickly sensation and
skin inammation resulting in the intra-epidermal retention
of sweat, such as occurs with miliaria rubra which has been
linked to elevated levels of IL-1 and IL-31detected in sweat.39
Therapeutic and Wellness Functions of Sweat
It is hypothesised that sweat produced by different activities
may differ in composition. For example, IL-1 concentrations
are increased in sweat induced by both exercise and sauna
bathing,39 yet exercise is linked to increases in the generation
of several end-metabolites like reactive oxygenated species
that are in turn linked to oxidative stresses. This is thought not
to be the case with sauna-induced sweat although this remains
to be validated by further studies.35
Lipid Homeostasis
Sebum production changes have been linked to diet. Caloric
deprivation in the setting of obesity decreases sebum
production while a high fat diet in the setting of psoriasis
increases it.67,68 Increases in energy intake have been associated
with increased excretion of triglycerides, cholesterol and
associated esters in sebum.35 As newer studies in sweat and
skin surface lipidomics are being done, more denitive
information regarding these links and potential mechanisms
of action are likely to emerge.69
Methods
Pubmed, Medline, Google Scholar, Embase, Science Direct,
Scopus, Ovid, Web of Science, Proquest, Toxline and UpToDate
databases were initially searched with keywords ‘sweat’ and
‘metabolomics’ with restrictions of English language and
of dates 2006–2016. These records were then supplemented
with searches for other research by key authors, searches of
citations and reference lists of key papers, and additional
searches with expanded keywords relating to sweat including
perspiration, sauna, exercise, secretion and/or excretion from
human skin and residual skin surface components as well
as expanded keywords relating to metabolomics including
exposomics, xenometabolomics, toxicometabolomics and
uxomics. Older studies of sweat (before 2006) have been
used in compiling background information, but not for the
detailed analysis of sweat collection methods.
Of the 1320 records identied for review as of 1 June 2016,
all 17 studies presenting quantitative human data utilising a
sweat metabolomics methodology of analysis between the
dates 2006–2016 were included, regardless of quality of
experimental design. An additional three sweat proteomics-
based studies were identied that utilised similar laboratory
platforms relevant to metabolomics and were also included in
the comparison analysis.
Results
The Table presents a summary of the pertinent information
regarding sweat induction and collection methods extracted
from the 20 identied studies for comparison.
Discussion
Sweat Induction Protocols
Induction of perspiration represents a phenomenon involving
a complex chain of metabolic reactions, with many possible
triggers, as already discussed. Exercise, temperature,
stress, psychological state, relative humidity, hormonal and
sympathetic/parasympathetic nervous system parameters,
diet, skin colonisation factors, xenobiotics exposure – both
purposeful and non-purposeful – can inuence sweat volumes
and content.40 Refer to the fourth column in the Table
describing sweat induction modes utilised in the reviewed
studies. A number of important factors are apparent when
obtaining sweat for metabolomics analysis: (i) ensuring
adequate amounts of sweat are available to complete the
analysis, including enough volume for controls and potential
further analysis; (ii) ensuring the mode of sweat induction
does not interfere with the utility of the results; and (iii)
ensuring that sweat induction and sweat collection happen
in a timely manner that optimises metabolic quenching and
metabolite stability.8,70
Pilocarpine Iontophoresis
Several active research groups rely on a chemical pilocarpine
iontophoresis method of inducing sweat.29,37,38,71,72 This
method takes advantage of the bioelectric properties of skin
which allow the application of low intensity electrical current
(i.e. 1.5 mA) for 5 min. The resulting opposition offered by
skin to this electrical current, called bio-impedance, is present
in intra-and extra-cellular uids and the capacitive reactance
of cell membranes. For a topically applied chemical such as
pilocarpine (0.5% pilocarpine nitrate solution), a drug with
cholinergic parasympathomimetic activity which aims to
stimulate primarily the muscarinic receptors of eccrine sweat
glands, to be absorbed through human skin, the electrical
current must overcome the bio-impedance imposed on its
ow to reach the target tissue of sweat glands with sufcient
intensity. This bio-impedance can be inuenced by a range of
factors, some of which are electricity source-dependent such
as the distance between electrodes positioning, pulsed direct
current vs constant direct current source, and size and content
of iontophoresis electrodes (typically containing 70% copper,
30% zinc with diameter of 30 mm).27,73
Some of the important host-dependent factors involved
with this mode of sweat induction include the amounts of
keratin and the variable thickness of stratum corneum (SC)
at different body sites, uctuating amounts of uid in skin
layers with overall hydration status, ambient temperature
increasing or decreasing hydration of keratin, adipose tissue
thickness (especially with some sweat glands residing in deep
dermis/ subcutaneous fat) and individual pain/tolerance to
the electric current. All of these factors can alter biological
responses, thereby potentially confounding metabolic results.
Therefore, the argument can be made that using pilocarpine
with iontophoresis induces production of a particular type of
primarily eccrine sweat but whether the detailed metabolomic
contents of this type of sweat are the same as physiologic
sweat and/or thermally-induced sweat and/or exercise-
induced sweat remains unknown.
After all, the original method of cholinergic stimulation
with pilocarpine iontophoresis on the skin to facilitate sweat
production dates back to the 1959 Gibson and Cooke publication
describing implementation and standardisation of the ‘classic
sweat test’ targeting sweat chloride levels for the purposes of
diagnosing cystic brosis (CF).74 The Webster Sweat Inducer
system coupled with a patented Macroduct Sweat Collector
used in more recent sweat metabolomics studies originates
from a further enhancement of the pilocarpine method, again
designed to specically improve the classic sweat test for
CF.75 The quantitative pilocarpine iontophoresis test (QPIT)
remains the gold standard for sweat induction in terms of
CF-related testing and now has over 50 years of progressive
standardisation.76 Despite better uniformity in collecting
sweat samples and improved reference intervals based on
age, dened rates of sweating and the volume of sweat to be
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 2322 Clin Biochem Rev 38 (1) 2017
Table. Sweat induction and collection methods for metabolomics.*
Study Aims n Sweat Induction Mode
Sweat Collection Sweat
Preparation
Protocols
Analytical Chemistry
Platforms
Methods Timing Amount Storage
Adewole et al.,
201672
Identify diagnostic
biomarkers of active
tuberculosis in eccrine sweat
83 Webster Sweat Inducer –
pilocarpine iontophoresis x
5 min
Macroduct® Sweat Collector – part of
Macroduct® Sweat Analysis System – covers
volar forearm x 15–35 min; sweat transferred
to micro-centrifuge tube
5 min induction
+ 15–35 min
collection
~10–30 µL Samples placed
on dry ice
immediately, stored
at -70°C until
analysed
Solubilised, reduced, alkylated, and digested
with protease; then dried, desalted, dried again,
then resuspended in ACN, formic acid
LC – MS/MS, untargeted
proteomics; FT mode for MS
detection, ion trap mode for
MS/MS detection
Jia et al., 201694 Assess feasibility of using
HPLC-MS/MS for accurate
quantifying of cortisol in
human eccrine sweat
4 Hot room set at 41°C and
~55% humidity
Leg skin cleansed with alcohol pads, followed
by dH2O and drying; sweat collected off skin
into Eppendorf LoBind micro-centrifuge tubes
25–30 min
collection times
>200 µL Samples placed
on dry ice
immediately, stored
at -80°C until
analysed
Sample mixed with ACN/ammonium acetate;
addition of internal standard (in ACN); ethyl
acetate extraction repeated twice, evaporated to
dryness, re-constituted in ACN
HPLC-MS/MS, SRM mode,
targeted
Sheng et al.,
201693
Monitor elimination of bio-
accumulated heavy metals in
humans with exercise
17 Exercise; no specic
instruction as to type or
location
Direct collection of sweat from any part of
the body into glass bottle with cover; then
transferred into 50 mL glass vials with lid.
Referenced methods from Genuis et al. 2011
utilised
Same day as
urine sample
collection
>20 mL Stored at -20°C
until analysis
Samples dried in oven for standardised weight,
ashed in furnace, cooled in dryer; residue
reconstituted in HNO3 with heat
Flame atomic absorption
spectrophotometry, targeted
Tang et al.,
201695
Compare levels of 5 heavy
metals (Cr, Cu, Zn, Cd, Pb) in
human sweat and urine after
physical exercise
9 Exercise; playing badminton
x 2 h
Upper bodies cleansed with ultrapure
H2O before exercise; sweat scraped into
polyethylene sample bottles. Samples allowed
to stand for 30 min, then ltered using 9-mm
lter paper into test tube
~2 h >20 mL Stored at 4°C until
testing
3 methods:
(i) direct dilution with HNO3
(ii) wet digestion with HNO3 + HClO4, heated
to 200°C; cooled, with HNO3 re-added, nal
dilution with ultrapure H2O
(iii) microwave digestion with HNO3 added,
microwaved, cooled, diluted with ultrapure H2O
ICP-MS, targeted
Delgado-
Povedano,
Calderon-
Santiago et al.,
201671
Develop and validate a
method for metabolomic
analysis of human sweat
using GC-TOF/MS
6 Webster Sweat Inducer –
Pilogel® Iontophoretic discs;
1.5 mA electric current x 5
min
Macroduct® Sweat Collector – part of
Macroduct® Sweat-Analysis System – covers
forearm skin x 15 min; sample transferred into
micro-Eppendorf tube
5 min induction
+ 15 min
collection
>70 µL each
participant –
pooled into one
sample
Frozen at -80°C Pooled sweat into each of 3 protocols:
(i) deproteinisation with methanol-ACN;
(ii) extraction with dichloro-methane;
(iii) extraction with ethyl acetate.
Each followed with methoxymation + silylation
GC-TOF/MS, full scan
mode, untargeted
Calderon-
Santiago et al.,
201538
Identify metabolic markers
of lung cancer in sweat to
develop screening tool for
diagnosis of lung cancer
96 Webster Sweat Inducer –
Pilogel® Iontophoretic discs;
1.5 mA electric current x 5
min
Macroduct® Sweat Collector – part of
Macroduct® Sweat Analysis System – covers
forearm skin x 15 min; sample transferred into
micro-Eppendorf tube
5 min induction
+ 15 min
collection
>10 µL Frozen at -80°C
until analysed
Diluted with formic acid and vortexed LC-QTOF MS/MS,
untargeted
Porucznik et
al., 201589
Targeted detection of BPA in
sweat in comparison to urine
for biomonitoring
50 Passive sampling – no
articial modes of sweat
induction
Sweat patches (PharmChek®) applied after
skin cleansed with alcohol wipes, to either
upper-outer arm or front/back midriff
7 days Not specied Sweat patches
stored and
transported in
sterile, BPA-
free 4-oz poly-
propylene
sample cups;
no temperature
specied
Sweat patches extracted with methanol;
evaporated in Turbovap®; reconstituted with
ammonium bicarbonate:
ACN (mobile phase)
UHPLC-MS-MS, targeted;
using methods initially
designed for urine samples
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 2524 Clin Biochem Rev 38 (1) 2017
Study Aims n Sweat Induction Mode
Sweat Collection Sweat
Preparation
Protocols
Analytical Chemistry
Platforms
Methods Timing Amount Storage
Dutkiewicz et
al., 201459
Untargeted metabolomics
proling of human sweat to
evaluate hydrogel micropatch
collection linked with direct
mass spectrometry
9 Passive sampling – in room
temperature, ~25°C, 45%
relative humidity
Skin pre-wiped with cellulose tissue soaked
with isopropanol: H2O; fabricated agarose
hydrogel micropatch embedded with PTFE
probe attached to forearm area with adhesive
bandage tape
1 min–1 h ‘single droplet’
– unable
to estimate
volume of
sweat sample
accurately
Hydrogel
micropatch probe
covered with glass
slide, stored at 4°C
Direct coupling of hydrogel micropatch probe
to nanospray desorption electrospray ionisation
mass spectrometer
ESI + IT + FT-ICR-MS
Calderon-
Santiago et al.,
201437
Untargeted global
metabolomics proling of
human sweat to optimise
laboratory methods and
chemometrics
96 Webster Sweat Inducer –
Pilogel® Iontophoretic discs;
1.5mA electric current x 5
min
Macroduct® Sweat Collector – part of
Macroduct® Sweat Analysis System – covers
forearm skin x 15 min; sample transferred into
micro-Eppendorf tube
5 min induction
+ 15 min
collection
>5μL Frozen at -80°C Pooled samples diluted with formic acid:H2O
with additional protocols:
(i) hydrolysis with 0.1M NaOH or HCL in H2O,
vortexed, evaporated to dryness, reconstituted
in chromatographic mobile phase A;
(ii) solid phase extraction using C18 and
hydrophilic centrifugal Micro SpinColumn™
LC-QTOF MS/MS,
untargeted
Shetage et al.,
201423
Identify collection methods
for RSSC and evaluate effects
of ethnicity, gender and age
on amount and composition
315 Passive sampling at room
temperature: 18–25°C, 50–
60% relative humidity
Forehead pre-wiped with cotton soaked in
diethyl ether, allowed to dry. Cigarette paper
applied, held in place with elastic headband, in
duplicate x 1 h, fresh cigarette paper replaced
every hour for total 3 h
3 h Totals not
specied:
peak amounts
0.11–0.12 +/-
0.06–0.07 mg/
cm2 of RSSC
collected in rst
hour
None specied Cigarette papers dehydrated x 2 h; extracted
with hexane; extract ltered through 0.2 micron
PTFE membrane, concentrated by purging
nitrogen
GC/MS, untargeted
Mark et al.,
201351
Detailed amino acid analysis
of sweat to better understand
key biological mechanisms
governing its composition
12 Hot room; 40°C, 60% relative
humidity x 15–40 min
Sweat droplets removed from axilla
with positive displacement pipette using
polypropylene tips; sample transferred directly
into ‘low binding’ Eppendorf tube kept at 4°C
~20 min >500 µL Frozen at -70°C Two methods:
i) ninhydrin derivatisation for amino acid
automated analyser
(ii )oximation and trimethyl-silylation for
GC-TOF/MS
Targeted amino acid analysis;
automated amino acid
analyser + GC-TOF/MS,
targeted
Raiszadeh et
al., 201229
Untargeted and targeted
analysis of healthy control
and schizophrenic patient
sweat, to identify candidate
biomarkers of disease
78 Webster Sweat Inducer –
pilocarpine iontophoresis
applied to volar forearm
Macroduct™ Sweat Collector – Macroduct
™ Sweat Stimulation and Sweat Collection
System (Elitech/WESCOR, Inc., Logan, UT,
USA); sample transferred into
micro-centrifuge tubes
30 min 50–60 µL Stored on dry ice Pooled samples:
reduction (dithio-threitol/urea), alkylation
(iodo-acetamide), overnight enzymatic
digestion (trypsin/ammonium bi-carbonate),
quenching (glacial acetic acid, then angiotensin
II), desalting (C-18 Zip Tips), drying in vacuum
concentrator, reconstitution in 0.1% formic acid
LC-MS/MS;
LC-MS/MS + spectral
counting;
MRM-MS verication
Genuis et al.,
201257
Targeted proling of phthalate
compounds in blood, sweat
and urine
20 Self-determined by
participants – infrared sauna,
steam sauna, exercise
Direct collection from any body site into 500
mL glass jar using stainless steel spatula;
participant-delivered to commercial laboratory;
transferred to 4 mL glass jars at laboratory
No time
parameters
around sweat
collection
except
conditional
within 1 week
of blood
collection
(before/after)
100 mL Stored at -20°C;
shipped frozen
on dry ice from
Canada to Sweden
for analysis
Not specied HPLC/MS, targeted;
GC/MS, targeted
Genuis et al.,
201255
Targeted proling of BPA in
blood, sweat and urine
LC-MS-MS, targeted
Genuis et al.,
201156
Targeted proling of 120
compounds (toxicants) in
blood, sweat and urine
ICP-MS, targeted
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 2726 Clin Biochem Rev 38 (1) 2017
collected at standardised sites as well as newer conrmatory
CFTR-based testing, there are still complicating factors.77-79
Documented reports of false positive and false negative sweat
chloride tests are in the literature, hypothesised to be due
to such wide ranging factors as contaminating topical gels,
interfering dermatological lesions (i.e. atopic dermatitis),
autonomic nervous system dysfunction, prostaglandin
use and other medication uses (e.g. topiramate), arsenic
toxicity, malnutrition states, immunoglobulin deciencies,
autoimmune disorders such as systemic lupus erythematosus,
and various abnormal endocrine states such as untreated
hypothyroidism and Addison’s Disease.27, 80, 81
Exercise/Sauna/Hot Rooms
Other forms of sweat induction used in the studies presented
in the Table include exercise and sauna activity or exposure
to elevated temperatures with varying humidity levels. Older
non-metabolomics studies have suggested distinct differences
in metabolic content when sweat is obtained from exercise or
sauna activity, especially in Ca2+ and Mg2+ concentrations.82
As this is an active area of ongoing research, it cannot be
assumed that metabolomics studies using exercise and/ or
sauna-produced sweat have interchangeable results. A further
potential confounder for sweat studies is the humidity level
of sauna or hot rooms as this may contaminate sweat samples
with condensation of airborne water droplets potentially
containing bacteria, viruses, fungi, and/or xenobiotics. This is
Study Aims n Sweat Induction Mode
Sweat Collection Sweat
Preparation
Protocols
Analytical Chemistry
Platforms
Methods Timing Amount Storage
Lee et al.,
201191
Untargeted metabolomics
analysis to determine
biochemical composition of
exercise sweat
48 Exercise on ergometer for 60
min
Collection with skin patch placed on the lower
back
Sweat patches
removed at
3 time-points:
10–20min,
30–40min,
50–60min of
exercise; placed
on dry ice
Not specied Frozen at -80°C
until analysis.
Not specied GC/MS and LC/MS/MS,
untargeted
Kutyshenko et
al., 201110
Untargeted metabolomics
analysis to determine
biochemical composition of
human sweat
10 Natural environmental heat Direct collection from forehead, upper chest,
upper/lower back, arms using glass pipette or
glass roller, rolled in tray with dH2O and/or
sterile spray gun lled with D2O sprayed
3–5 min
collection
+ 7–10 min
sample
preparation
>0.56 mL Sample storage not
specied; analysis
performed 10–15
min after sweat
collection
Diluted with D2O, centrifuged, transferred to
standard NMR tube
1H NMR Spectroscopy
– high resolution, both
one dimensional and two
dimensional, untargeted
Michael-Jubeli
et al., 201169
Develop simple analytical
protocol for qualitative
characterisation of individual
SSLs and quantitative
evaluation of lipid classes
1 Passive sampling Lipid-free absorbent papers placed on 6 areas
– forehead, back, thorax, forearm, thigh, calf
– maintained for 30 min with medical tape;
removed with tweezers and placed into closed
vials. Collections repeated 4 times
30 min
collection
Not specied Storage of
unprocessed
samples not
specied
Extracted with diethyl ether twice, concentrated
with rotary evaporation, transferred into
2 mL vials, dried under nitrogen stream;
dried extract stored at -20°C until analysis;
extracts derivatised/trimethyl-silylated; rotary
evaporated, residue dissolved in isooctane
HTGC-MS, with electron
impact and chemical
ionisation
Penn et al.,
200792
Test the validity of individual
odour hypothesis by analysing
VOCs in sweat, urine and
saliva
197 Passive sampling Axillary sweat sampled with devised twister
PDMS-coated stir bars, held by special rollers,
placed directly on skin; samples transferred to
glass vials
Once each
fortnight
sampling over
10-week period;
unspecied
sweat collection
timing
Not specied Stored at ~4°C;
shipped in cooler
each week from
Austria to USA for
analysis
Samples directly analysed with SBSE in
connection with thermal desorption GC-MS
SBSE with thermal
desorption GC-MS
Harker et al.,
200628
Untargeted metabolomics
analysis of human eccrine
sweat
60 Hot room at 43.3°C, 65%
relative humidity x 15–40 min
Underarm area wiped, then sweat collected
with plastic-tipped pipette, sample transferred
into sealed glass vials
15 min
collection
>50µL Frozen at -20°C
until analysis
Samples diluted and deuterated phosphate
buffer (pH 7.4, 0.1M); transferred into 5 mm
OK NMR tubes
1NMR Spectroscopy – high
resolution, one dimensional,
untargeted
BPA – Bisphenol A; PTFE – polytetra-uoro-ethylene; PDMS – polydimethyl-siloxane; RSCC – residual skin surface components; SSLs – surface skin lipids; VOCs – volatile organic compounds; ACN – acetonitrile; SBSE – stir bar sorptive extraction; LC-MS/MS – Liquid
Chromatography-Tandem Mass Spectrometry; HPLC-MS/MS – High Performance Liquid Chromatography-Tandem Mass Spectrometry; SRM – selected reaction monitoring; ICP-MS – Inductively Coupled Plasma mass Spectrometry; GC-TOF/MS – Gas Chromatography-
Time of Flight/ Mass Spectrometry; LC-QTOF MS/MS – Liquid Chromatography-Quadripole Time Of Flight-Tandem Mass Spectrometry; ESI – Electrospray Ionisation; IT – Ion Trap; FT-ICR-MS – Fourier Transform Ion Cyclotron Resonance mass spectrometry; MRM-
MS – Multiple Reaction Monitoring-Mass Spectrometry; UHPLC-MS-MS – Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry; GC / MS – Gas Chromatography / Mass Spectrometry; HTGC-MS – High Temperature Gas Chromatography-Mass
Spectrometry; 1H NMR – Proton (Hydrogen -1 nuclei) Nuclear Magnetic Resonance Spectroscopy; ICP-MS – Inductively Coupled Plasma Mass Spectrometry; PTFE – polytetrauoroethylene; dH20 – distilled water; -D2O – deuterated, heavy water
*See Appendix (online supplement) for an expanded version of this table including more detailed information of sweat preparation protocols, chemometrics, databases and key ndings pertaining to studies.
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 2928 Clin Biochem Rev 38 (1) 2017
especially the case if direct analysis of sweat without detailed
cleaning and extraction methods is employed. There is also a
documented progressive decline in sweat rates when the skin
is thoroughly wetted and/or with higher humidity conditions,
referred to as hidromeiosis. This can be followed by an after-
effect of increased sweating when the skin is outside the
exposed high humidity environment. The timing and rate of
sweating therefore depends upon ambient temperature and
humidity.83
Passive Sampling/Physiological Sweating
Some of the studies in the Table used only passive sampling
of sweat without any imposed modes of sweat induction.
Most of the time, longer periods of sweat collection were
deemed necessary to obtain the same or smaller amounts of
sweat or residual skin surface components (RSCCs). This
may compromise the results, especially in terms of metabolic
quenching of enzymatic reactions and metabolite stability.
With untargeted metabolomics analysis, this is highly relevant
due to unknown metabolites and thus unknown metabolite
stability proles. With more targeted metabolomics, as in
the case of sweat testing for various drugs of abuse, this
would depend on desired levels of quantication and the
relative stability of the target molecule in certain specied
conditions, necessitating controlled stability studies to be
done, as discussed more extensively in a recent review by
de Giovanni and Fucci.1 With newer technological trends
of sweat analysis requiring smaller amounts of sweat, the
need for complicated sweat induction methods will likely be
reduced or redundant.6,59,84,85
Sweat Collection Protocols
A brief overview of the sweat collection methods in the Table
reveals a diverse range of techniques employed to collect sweat
for metabolomics studies. These techniques vary from simple,
direct collection of sweat off skin into microcentrifuge tubes
or glass jars, to elaborate specically-designed implements
(i.e. glass pipettes and rollers, hydrogel micropatches) to
more commercially-available products like the Macroduct®
Sweat Collector or PharmChek® sweat patches. Large
variations between individuals in the amounts and location of
sweat produced create major difculties for those attempting
to design a universal sweat collection device. Skin irritation,
alterations of skin pH, disruptions to skin barrier properties
and interactions with differing individuals’ skin microbiota
are just some of the difculties to be encountered in designing
an ideal sweat collecting apparatus.1
Commercial Sweat Collection Devices
The Macroduct (ELITECH Wescor® Inc., Logan, UT, USA)
is a popular commercially-available sweat collector that
employs a plastic capillary-coil device of 29 mm diameter to
wick the sweat off the skin surface, usually of the forearms.86
Since its introduction in 1986, it has been used in several
sweat studies, including several addressing sweat metabolome
optimisation.37,71 The Macroduct® is a component of the
Macroduct® Sweat Analysis system, involving a commercial
apparatus covering the skin after iontophoresis stimulation
using pilocarpine29,37,38,71,72 that was developed to reduce
the problems encountered with older lter pad- and tissue
paper-based sweat collections of sweat chloride testing for
CF.86 Macroduct® helps to overcome issues of background
contamination, encapsulation (which increases local skin
temperature and sweat gland secretion) and hidromeiosis
(the progressive decline in sweat rates that occurs when
skin is thoroughly wetted and/or with higher humidity)
encountered with the older methods.86 The Macroduct® has
a capacity of ~0.1 mL of sweat collection per device session.
Some researchers have considered placing more than one
Macroduct® simultaneously to collect larger amounts of
sweat that are then pooled for analysis, but differing sweat
rates at different collection sites, amplication of inconsistent
dilutional effects and difculty in attaching the Macroduct®
to other body sites produces confounding results.29
The same corporation (ELITECH Wescor®, Inc., Logan,
UT, USA) developed a larger version of the Macroduct®
called the Megaduct®. This is a round, plastic concave-based
device with a larger collection area of 22.1 cm2 and a central
aperture through which sweat collects into coiled capillary
tubing. While the Megaduct® has an increased sweat volume
capacity of ~0.5 mL,40,87 its utility is limited by the duration of
heat and/or exercise necessary to sweat long enough to ll the
Megaduct® reservoir. For example, in one study, it required
65–75 min to collect the full 0.5 mL reservoir of sweat in 10
healthy men, with varying exercise intensities (VO2 = 0.5–2.0
L/min), temperatures (20–40 °C) in a controlled 50% humidity
environment.87 Increasing sweat collection times to this range
(>60 min) can potentially impact the power of metabolic
ndings, especially with the issues of metabolic quenching
and time course of metabolic changes.87 For example, it is
known that concentrations of sweat electrolytes and minerals
such as zinc and iron change in relatively short periods of
time (<30 min).87,88 Furthermore, both the Macroduct® and
Megaduct® are designed primarily for forearm placement. As
discussed already, the human body does not have a uniform
sweat rate or composition over all skin locations. In fact,
results of one study suggest forearm sweat rate is 30–60%
less than that of the chest or back.87
Another popular commercial sweat collection device is the
PharmCheck® or PharmChek® (PharmChem Inc., Fort
Worth, TX, USA) sweat patch which has been available
since 1990 (refer to the sweat collection method used by
Porucznik et al., 2015 in the Table).89 This device is a non-
occlusive patch consisting of a medical-grade cellulose paper
absorption pad covered by a thin layer of polyurethane and
acrylate adhesive. To use it, the skin site must rst be cleaned
with a tolerable solvent (e.g. isopropyl alcohol swabs) and
thoroughly dried before application. The adhesive lm of the
patch is a semipermeable barrier that allows oxygen, carbon
dioxide and water (vaporised by body heat) to diffuse freely.
Larger non-volatile molecules (such as drugs, metabolites,
metals and other xenobiotics) are retained on the inert
cellulose absorption pad of the patch. Contaminants from
the environment cannot penetrate the adhesive barrier from
the outside once it is in place, enabling the patch to be worn
during normal activities, including bathing, swimming and
other exercise. It has a release liner that allows removal of
the collection pad only once from the adhesive layer after
use thereby preventing removal, reapplication or tampering
with the patch. Underneath the polyurethane layer is a unique
9-digit number printed on the patch that is visible through a
purpose-made window for legal (or research) applications.
These features make this device useful in sweat testing for
illicit drugs.1
Some of the disadvantages of PharmChek® include high
inter-subject variability (potentially due to variable body
site placement), high cost, possibility of environmental
contamination either before patch application or after patch
removal, risk of accidental removal before desired monitoring
period and differing rates of drug/metabolite/xenobiotic
penetration through the membrane, depending upon charged
or uncharged state. Molecules in an uncharged state have
been recorded to migrate more rapidly than charged species
in studies of PharmChek®.1,90
Non-Commercial Sweat Collection Techniques
A newer form of sweat patch with commercial potential
described by Dutkiewicz et al. is a specically-designed
agarose hydrogel micropatch with polytetrauoroethylene
(PTFE) support that has been developed for simplied
collection of very small amounts of sweat that can be analysed
directly within minutes using various MS platforms.59
This new method of sweat collection shows promise, but
still requires further validation and optimisation of signal
sensitivity and performance at higher temperatures and at
increased sweat rates.59
Other noncommercial techniques of sweat collection for
metabolomics studies are also documented in the Table. Lee
et al. describe a ‘sweat collection patch’ placed on the lower
back with sweat collected at three time points (10–20 min,
30–40 min, 50–60 min) while participants exercised on a
cycling ergometer.91 Sweat was frozen on dry ice, and then
stored at –80 °C until prepared and analysed. Unfortunately,
there is limited mention of skin preparation, the type of sweat
collection patch used, how the sweat is frozen, either intact
in patch or transferred to another collection tube, or how
sweat is prepared for untargeted metabolomics analysis.91
Occlusive skin patches consisting of 2–3 layers of lter paper
or gauze have been used in other sweat collection studies
but limitations of excessive pH variations and skin irritation
with some degree of presumed skin disruption have been
signicant detractors.40
Shetage et al. and Michael-Jubeli et al. both use passive
sampling with ‘cigarette paper’ and ‘lipid-free absorbent paper’
to collect the desired RSCCs or surface skin lipids (SSLs),
respectively. These collection methods have advantages of
economics and simplicity but still have the disadvantages of
encapsulation and hidromeiosis already discussed as well as
long collection times of 3 h and 30 min respectively.23,69
Kutyshenko et al. describe specially-designed glass rollers
and glass pipettes for sweat collection. The rollers were used
on lower sweat-producing regions (e.g. arms) moisturised
with a sterile distilled water spray gun beforehand whilst the
glass pipettes were used on heavier sweat-producing areas,
namely forehead, chest and back.10 Although the use of glass
is compelling with its relative inertness and is certainly of
benet when metabolomically targeting plastics-related
xenobiotics, the confounders of varied locations of sweat
harvesting, dilutional effects of adding sprayed distilled water
and a lack of standardisation of temperature and humidity are
likely to complicate the untargeted ndings of this study.
Penn et al. describe another unique, specially-designed
method of collecting sweat with a polydimethylsiloxane-
coated stir bar that is rolled directly onto skin. The fact that
sweat samples can then directly go through the necessary
extraction step with a thermal desorption GC-MS setup is
attractive. However, the fact that samples had to be shipped
at 4 °C overseas to a special laboratory is a limitation and
raises the issues of sample contamination and metabolite
degradation during transportation.92
Unsupervised Sweat Collection Techniques
In studies by Genuis et al. and Sheng et al., participants were
instructed to collect perspiration from any site on their body
directly into a laboratory-provided, pre-cleaned, acid- and
water-rinsed 500 mL glass jar or by using a stainless steel
spatula against their skin to transfer perspiration directly
into the same laboratory glass jar.55-57,93 Sweat was collected
within one week before or after specied blood collection and
participants delivered the collected sweat sample themselves
to a laboratory without any specied storage or transport
Sweat Collection for Metabolomics AnalysisHussain JN et al.
Clin Biochem Rev 38 (1) 2017 3130 Clin Biochem Rev 38 (1) 2017
timeframes. The choice of glass for storage container concurs
with the previous study discussed above. Including the option
of stainless steel spatulas for sweat collection is intriguing.
A grade of stainless steel was reportedly chosen to match
the composition of laboratory needles used in standard
blood collections since sweat was being directly compared
to similarly-targeted detections of compounds in blood and
urine in these studies. Recognising that stainless steel contains
varying amounts of primarily iron, nickel and chromium that
can also be found in trace amounts of physiological sweat, this
is indeed important to factor in with future sweat metabolomics
studies.56 However, the relative absence of controlled timing,
temperature, humidity and storage conditions of sweat
samples and the presumed delay of a metabolic quenching
step make the sweat collection protocol of these series of
studies less than ideal for metabolomics studies.
Direct Sweat Sampling Techniques
In a yet different approach, Harker et al. detailed subjects
rinsing and drying their axillae with water just before entering
a hot room (set at 43.3 °C and 65% relative humidity) for
15–40 min.28 Subsequently, the underarms were ‘wiped’ in an
unspecied way and sweat was collected with a plastic-tipped
pipette and transferred directly into glass vials with a collection
period of approximately 15 min. The glass vials were then
immediately sealed and stored frozen at –20 °C until analysis.
This method included several commendable presampling
controls by limiting use of pharmaceutical medications and
topical applications of antiperspirant and soap products,
detailing dietary limitations and specifying shaving of the
axillary hair. However, the wiping of underarms immediately
before sweat collection introduces potential issues of altered
skin integrity on a molecular level which may impact the
content of sweat analysis with 1H NMR spectroscopy. As
mentioned in the section discussing sweat functions, the skin
integrity is thought to inuence pathways of water and other
molecules/metabolites either via transmembrane proteins or
lipid membranes or sweat glands during uid transport from
plasma to skin surface. Not specifying the exact material the
underarms were wiped with and mixing plastic with glass
in the sweat collection and storage before analysis create
additional uncertainty.28
Some of the researchers associated with the Harker et al.
study went on to publish another study of axillary sweat with
similar direct sweat collection techniques, but optimised and
targeted for amino acid analysis with a different metabolomics
platform of GC-TOF (Time of Flight)/MS instead of 1H NMR
spectroscopy.51 While direct sampling with minimal handling
time and prompt metabolic quenching are advantages of this
method, wiping the armpit before sampling and the use of
a positive displacement pipette that might disrupt the skin
surface along with the requirement for trained personnel to
perform the sampling task remain as drawbacks.51 The choice
of harvesting sweat from the axillae, rich in apocrine and
apoeccrine sweat as well as eccrine sweat, in both of these
studies complicates the comparisons to be made with other
metabolomics studies harvesting sweat from other specic
areas of the body with minimal apocrine or apoeccrine
contributions.
Some similar advantages and disadvantages are appreciated
with the sweat collection methods of Jia et al.94 Leg skin
cleansing with alcohol pads followed by distilled water
rinsing and drying precede the direct collection of sweat into
microcentrifuge tubes which were placed immediately on dry
ice to effect metabolic quenching.94 The cleansing and rinsing
beforehand, as well as the physical, direct contact with the
microcentrifuge tube may however disrupt the skin surface
and again potentially alter skin integrity with its possible
effects on uid migrations from plasma to skin surface.
Summary
A diverse range of sweat induction modes and sweat
collection methods are presented in the Table, all with
their own advantages and disadvantages. Issues of variable
location, timing and amounts of sweat induction and sampling
as well as inconsistent sample processing steps and storage
conditions confound most comparisons between methods.
Optimising these parameters and exploring newer identied
concepts surrounding sweat collection based upon updated
information about sweat glands and the collective contents
of their secretions will generate more meaningful results to
build and improve our knowledge of the sweat metabolome.
Standard operating protocols (SOPs) for collecting human
biouids like urine, blood and sweat for metabolomics studies
are crucial to help control for the wide variety of factors that
can inuence metabolite concentrations. The SOPs for human
sweat collection require updating beyond cystic brosis and
illicit drug testing models to optimise metabolomics results.
The following considerations need attention in future studies:
• Specifying body sites of human sweat collection is
of utmost importance in future comparisons of both
targeted and untargeted metabolomics studies. Not all
sweat collected anywhere on the body can be assumed
homogenous in metabolic content.
• Until further comparative studies are done, consideration
should be given to subclassifying sweat based upon
induction approaches – i.e. pilocarpine-induced sweat
vs physiological sweat vs thermally-induced sweat vs
exercise-induced sweat.
• Measures to ensure adequate skin integrity other than
mere visual inspection at sweat collection sites need
further development and study.
• Examining the molecular content of sweat induction,
collection and storage devices for potential adsorption
and metabolic reactivity requires further attention and
attempts at standardisation.
• Minimising the timing of sweat collection, transport
and storage as well as ensuring a timely and adequate
metabolic quenching step is important for comparing
future sweat metabolome studies.
• Environmental factors of temperature and humidity
signicantly impact the metabolic parameters of sweat and
need to be specied and ideally standardised. Furthermore,
the potential inter-relationship between overall core body
temperature and local skin temperature at sweat collection
sites may impact sweat metabolomics results.
• Attention to controlling individual variables such as
diet content, fasting vs postprandial state, exercise state,
emotional state, pharmaceutical and/or recreational drug/
supplement use and underlying medical conditions that
could impact the pH or overall metabolic state is important
when interpreting any metabolomics results. This applies
to controls instituted both preceding and during collection
of sweat.
• Dening the minimum amounts of sweat necessary to
overcome intra-individual and inter-individual global
metabolomic differences, stretching beyond guidelines
based upon CF-specic testing of pilocarpine-induced
sweat, is still a work in progress. Clarications between
physiological sweating and exercise- or thermally-induced
sweating within this context are also necessary.
• Age-specic inuences on sweat metabolomics results
will require further investigation.
• Attempts at simultaneously characterising the individual
skin microbiota (colonising bacteria, viruses, fungi, etc.)
both quantitatively and qualitatively at sites of sweat
collection might further elucidate suspected important
metabolic relationships.
Conclusion
Better standardising of human sweat induction and collection
methods to address the important challenges identied in this
review is a key step to furthering sweat metabolomics. If this
can be achieved, it is anticipated that sweat may become a
more utilised biouid capable of delivering easily accessible,
individualised and instantaneously useful metabolic
information that signicantly enhances our knowledge of
human health and disease.
Acknowledgements: This manuscript was developed as
part of study conducted by Dr Joy Hussain during her PhD
candidature. We wish to thank the Jacka Foundation of
Natural Therapies for providing an academic scholarship to
support her candidature and therefore this study.
Competing Interests: None declared.
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