Relationships between electrolyte and amino acid compositions in sweat during exercise suggest a role for amino acids and K+ in reabsorption of Na+ and Cl- from sweat.

Concentrations of free amino acids and [K+] in human sweat can be many times higher than in plasma. Conversely, [Na+] and [Cl-] in sweat are hypotonic to plasma. It was hypothesised that the amino acids and K+ were directly or indirectly associated with the resorption of Na+ and Cl- in the sweat duct. The implication would be that, as resources of these components became limiting during prolonged exercise then the capacity to resorb [Na+] and [Cl-] would diminish, resulting in progressively higher levels in sweat. If this were the case, then [Na+] and [Cl-] in sweat would have inverse relationships with [K+] and the amino acids during exercise. Forearm sweat was collected from 11 recreational athletes at regular intervals during a prolonged period of cycling exercise after 15, 25, 35, 45, 55 and 65 minutes. The subjects also provided passive sweat samples via 15 minutes of thermal stimulation. The sweat samples were analysed for concentrations of amino acids, Na+, Cl-, K+, Mg2+ and Ca2+. The exercise sweat had a total amino acid concentration of 6.4 ± 1.2mM after 15 minutes which was lower than the passive sweat concentration at 11.6 ± 0.8mM (p<0.05) and showed an altered array of electrolytes, indicating that exercise stimulated a change in sweat composition. During the exercise period, [Na+] in sweat increased from 23.3 ± 3.0mM to 34.6 ± 2.4mM (p<0.01) over 65 minutes whilst the total concentrations of amino acids in sweat decreased from 6.4 ± 1.2mM to 3.6 ± 0.5mM. [Na+] showed significant negative correlations with the concentrations of total amino acids (r = -0.97, p<0.05), K+ (r = -0.93, p<0.05) and Ca2+ (r = -0.83, p<0.05) in sweat. The results supported the hypothesis that amino acids and K+, as well as Ca2+, were associated with resorption of Na+ and Cl-.

Introduction

It has been well documented that eccrine sweat produced for evaporative cooling in humans contains water, electrolytes, lactate, ammonia and urea [1-3]. The electrolytes predominantly consist of Na+ and Cl- and to a lesser extent, K+, Mg+ and Ca+. Sweat also contains an array of free amino acids including serine, histidine, ornithine, alanine, glycine and lysine that are present at concentrations several times higher than their corresponding levels in plasma [4-6]. Little is known about the function of free amino acids in sweat or why they do not mirror the relative plasma compositions. It has been proposed that at least some of the relative increases of certain amino acids such as serine, histidine and glutamic acid relates to their function as humectants in the natural moisturising factor (NMF) of the outer layers of the skin [5, 7, 8]. However, small contributions from the NMF would be unlikely to account for the >ten-fold increases in concentration observed in sweat relative to plasma[7].

Estimates of the number of eccrine sweat glands on the human body range between 1.6–4 x 106, with the highest densities located on the palms, finger print ridges and the soles of the feet [9]. It has previously been thought that the primary source of sweat electrolytes was derived from blood plasma filtrate of serous secretion through epithelia that have low transepithelial resistance [3, 10, 11]. More recent perspectives suggest, however, that sweat is a “constructed fluid” primarily under cholinergic control in humans; the process begins with active pumping of Cl- followed by Na+ into the secretory coil to a high osmolality which in turn draws water into the lumen to re-equilibrate [2]. In this way, the preliminary sweat fluid produced by the eccrine secretory coil contains Na+ and Cl- levels isotonic to plasma [3, 12]. This preliminary fluid is pumped along the duct where Na+ and Cl- are then actively resorbed to varying degrees depending on the body location [3, 13].

Ductal resorption of Na+ and Cl- has been attributed to the epithelial sodium channel (ENaC) and cystic fibrosis transmembrane regulator (CFTR) channel, occurring when Na+ is pumped through the ENaC and Cl- follows passively. This is made possible as the loss of Na+ from the lumen of the duct creates an electrochemical gradient allowing Cl- to enter the cell via the CFTR channels [14] which are ATP-dependent and pH sensitive [15]. Additional Cl- in the lumen is exchanged for HCO3- via anion exchangers embedded in the luminal cell membrane [16]. The sweat released from the duct of the eccrine sweat glands contains Na+ and Cl- ions at 10–30% of the concentration at which they occur in the plasma [17, 18]. In contrast, the level of K+ in the final sweat is usually 2–3 times higher than found in plasma [3]. It has been suggested that secretion of K+ into the sweat fluid within the duct may occur as a result of the active resorption of Na+ but this is not fully understood [2]. Electrolyte resorption in the duct is important in avoiding exercise-associated hyponatremia (EAH), which occurs when the blood [Na+] falls below 135 mmol/L [19]. It commonly occurs in endurance athletes due to a combination of hypotonic fluid intake, reduced or no intake of Na+ from food and losses of Na+ from sweat and urine [20]. Concentrations below 120 mmol/L can result in fatigue, nausea, acute vomiting, acute epileptic seizures and neurological deficits [21].

Serine, histidine, alanine and ornithine are generally found at high levels in sweat compared with corresponding levels in plasma whilst glutamine and proline have been found at relatively low levels [4-7]. To date, little explanation for the high concentrations of amino acids in sweat has been proposed and there is no understanding of why glutamine and proline are selectively conserved. Shaping a detailed model of the cellular mechanisms that contribute to the high concentrations of amino acids in sweat is integral to determining the impacts of sweat facilitated losses of amino acids. Previous research has indicated that amino acid content in sweat decreased over the course of exercise [7]. An earlier study that collected and analysed sweat samples from the lower back of four athletes during exercise noted that [Na+] increased gradually for the first 40 minutes of exercise before plateauing at 26.2 mM (± 19.4 mM) [22]. The physiological impacts of increased Na+ losses in sweat with time and diminished levels of amino acids may be linked with, and represent a mechanism for, heat adaptation.

Since the concentrations of certain amino acids and K+ have been reported at significantly higher concentrations in sweat than in plasma [6, 23, 24], it was hypothesized that these components could be either directly or indirectly involved with the process of resorption of Na+ and Cl- in the sweat duct. Alterations in the sweat concentrations of Mg2+ and Ca2+ could also potentially contribute to the resorption processes. It was thus proposed that if amino acids, K+, Mg2+ and Ca2+ were involved in the resorption of Na+ and Cl-, then [Na+] and [Cl-] would be inversely correlated with concentrations of these sweat components over the duration of the exercise period. The aim of the current study was to investigate potential relationships between [Na+] and [Cl-] with [K+], [Mg2+], [Ca2+] and the amino acids in sweat collected from recreational athletes over a defined period of cycling exercise. The influence of exercise on sweat composition was also tested by comparisons with sweat collected under passive conditions of thermal stimulation.

Materials and methods

Ethics statement

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Eleven recreational athletes were recruited from the University of Newcastle’s Ourimbah and Callaghan campuses to participate in a 65-minute cycling session for sweat collection (seven males (weight 85 ±9 Kg, height 1.8 ± 0.05 m) and four females (weight 65 ± 7, height 1.7 ± 0.05 m), with an age of 28 ± 10y. The group undertook an average of 5.2 hours of exercise per week in the two weeks prior to the study (SD ±2.65 hours, range 2–10.5 hours). This research was approved by the University of Newcastle Human Research Ethics Committee (approval number: H-2014-0086) and the participants provided written informed consent prior to inclusion in the study.

All athletes provided a passive sweat sample from a pre-washed forearm by sitting in a heated tent area at 32°C (±0.4°C) for up to 15 minutes. All participants had their forearms washed with mild detergent and warm water up to the elbow, which was then dried with a clean paper towel. The collected sweat was aspirated into a Monovette™ and stored at 4°C until analysed. However, only six of the eleven participants produced an adequate sweat sample during the passive sweat collection.

The participants underwent a YMCA Sub Maximal Cycle Ergometer test to determine maximum predicted workload [25, 26]. A plastic bag was sealed around one arm of each participant just below the elbow to collect sweat. The participants rode on a Monark cycle ergometer at 32°C (±0.4°C) and 42–57% relative humidity at 60% predicted VO2 max for a total of 65 minutes. Sweat samples were collected following 15, 25, 35, 45, 55 and 65 minutes of exercise. The participant would dismount the cycle ergometer, the bag was removed, each participant’s arm was re-washed and sealed with a clean bag. The sweat collected in the bag was transferred to a Monovette™ transport tube which was then stored at 4°C until analysis.

Amino acids in the sweat samples were extracted and processed with a Phenomenex® EZ:faast™ sample kit for analyses by gas chromatography flame ionisation detection (GC-FID) as described previously [7]. A Zebron® 50% phenylpolysiloxane, 50% Methylpolysiloxane column (ZB-50) with dimensions 10 m x 0.25 mm and 0.25 μm film thickness, was used to run all samples. A pulsed splitless method was used with a starting temperature of 150°C which increased at 32°C per minute to a holding temperature of 320°C. The flow rate was 2 mL/ minute and the carrier gas was Helium. The run time was 7.64 minutes in total. All GC-FID operations were controlled through Agilent® ChemStationTM software which had been calibrated for each amino acid with standard concentrations of 5, 10, 20, 30 and 40 nm/100 μL against corresponding FID signal peak area. Norvaline (20 nm/100 μL) was added at the beginning of each sample preparation as the internal standard. The amino acids able to be integrated by this method included L-alanine, L-sarcosine, L-glycine, α-aminobutyric acid, L-valine, ß-aminoisobutyric acid, L-leucine, L-alloisoleucine, L-isoleucine, L-threonine, L-serine, L-proline, L-asparagine, L-thioproline, L-aspartic acid, L-methionine, L-hydroxyproline, L-glutamic acid, L-phenylalanine, α-aminoadipic acid, α-aminopimelic acid, L-glutamine, L-ornithine, glycyl-proline (dipeptide), L-lysine, L-histidine, L-hydroxylysine, L-tyrosine, proline-hydroxyproline (dipeptide), L-tryptophan, L-cystathionine and L-cystine. Total amino acid concentrations were based on summation of the measured individual amino acids.

To measure Na+, Mg2+, K+ and Ca2+ concentrations in sweat, samples were diluted 1:100 in 2% NHO3 in a Labcon® MetalFree 15 mL tube and mixed thoroughly for 30 seconds. The samples were left to digest for 48–72 hours before 10 μL of an Agilent internal standard mix containing 100 ppm of Li6, Sc, Ge, In, Lu, Rh, Tb and Bi was added to each sample to produce a final concentration of 100 ppb. Each sample was run on an Agilent ICP-MS 7900. To determine Na+, Mg2+, K+ and Ca2+ concentrations in each sample, High Purity Standards™ ICP-AM-15-1M mix was used to make calibration standards of concentrations 0, 1, 5, 10, 50, 100, 500, 1000, 5000 and 10000 ppb. These were prepared freshly and run immediately prior to each batch of sweat samples to produce a 10 point calibration curve employed upon batch completion by Agilent Technologies MassHunter 4.2 Workstation software to quantify [Na+]. [Cl-] was determined in the same way with an 8 point calibration curve (0 ppb, 0.606 ppb, 6.06 ppb, 60.6 ppb, 0.606 ppm, 6.06 ppm, 15.15 ppm and 30.3 ppm) prepared from a 10, 000 ppm stock solution of Fluka NaCl in Milli Q water. The internal standard mix was added to both calibration standards making a final concentration of 100 ppb.

Electrolyte and amino acid concentrations across the exercise protocol were analysed using one-way repeated measures ANOVAs. Upon significant time effects, further analysis were performed using Duncan’s multiple range test. Additionally, Pearson’s correlations were performed to assess interrelationships between outcome variables. All statistical analyses were conducted using TIBCO Software Inc. (2017), Statistica (data analysis software system), version 13 and statistical significance was accepted at p<0.05.

Results

The evaluations of electrolytes and amino acids in sweat have been summarised in Table 1 with reference to comparisons with literature values for plasma composition (electrolytes [27] and [28]; plasma [29]). [Na+] and [Cl-] were present in the passive sweat at 10% of the plasma concentrations, [Mg2+] at 22% and [Ca2+] at 34%, whereas [K+] was two times more concentrated than in plasma. The average total level of amino acids was nearly four times higher in the passive sweat compared with the literature values for plasma. Serine, glycine, alanine, ornithine and aspartic acid were the most abundant amino acid components in the sweat. Aspartic acid was 100 times more concentrated in the passive sweat than plasma and serine, ornithine and glycine were 26-fold, 14-fold and 7.4-fold higher than the corresponding literature plasma levels. Only trace levels of glutamine were detected in the passive sweat (Table 1).

Table 1

The average concentrations of the electrolytes and amino acids in sweat collected from the recreational athletes were measured at six time points over the 65-minute period of exercise for comparison with literature plasma concentrations.

Passive sweat samples were collected from six participants.

Electrolyte and amino acid concentrations (μM)	Passive sweat(n = 6)	Literature PlasmaμM ± SE	Exercise sweat (n = 11)						Correlations	Na+	Cl-	K+	Mg2+	Ca2+
			15 min	25 min	35 min	45 min	55 min	65 min
Na+	14,385 ± 1226	140,400 ± 500	23,275 ± 3,053	28,703 ± 3,146	30,481 ± 2,950	30,488 ± 2,220	32,291 ± 2445	34,598 ± 2,366^	Na+		0.99	-0.93	-0.65	-0.83
Cl-	10,910 ± 1945	104,500 ± 500	17,163 ± 4,293	22,157 ± 3,922	25,335 ± 3,758	24,840 ± 2,031	25,573 ± 2789	28,595 ± 3,105	Cl-			-0.93	-0.65	-0.83
K+	10,358 ± 685	4,420 ± 80	8,224 ± 929	6,250 ± 600	5,908 ± 639	5,517 ± 631	5,548 ± 625	5,489 ± 503	K+				0.82	0.96
Mg2+	211 ± 26	964 ± 13	131 ± 24*	91 ± 15	93 ± 14	98 ± 14	100 ± 12	104 ± 15	Mg2+					0.95
Ca2+	840 ± 95	2,483 ± 14	555 ± 75*	324 ± 43	331 ± 44	313 ± 33	331 ± 33	333 ± 35^
Serine	3,030 ± 226	114	1,835 ± 336*	1,454 ± 285	1,217 ± 267	1,241 ± 193	1,013 ± 186	1,004 ± 203	Serine	-0.98	-0.97	0.94	0.64	0.82
Glycine	1,751 ± 150	236	997 ± 202*	804 ± 169	689 ± 154	700 ± 120	580 ± 99	595 ± 87	Glycine	-0.97	-0.96	0.94	0.66	0.83
Alanine	958 ± 96	419	601 ± 123	475 ± 102	426 ± 100	429 ± 79	342 ± 63	348 ± 53	Alanine	-0.98	-0.95	0.93	0.64	0.82
Ornithine	928 ± 144	65	452 ± 85*	327 ± 60	272 ± 50	289 ± 43	273 ± 36	291 ± 40	Ornithine	-0.89	-0.90	0.97	0.86	0.95
Aspartic acid	702 ± 72	7	415 ± 99	299 ± 61	281 ± 60	277 ± 47	244 ± 41	245 ± 34	Aspartic acid	-0.97	-0.95	0.98	0.79	0.92
Threonine	661 ± 45	146	412 ± 77	319 ± 61	277 ± 60	283 ± 43	216 ± 34	211 ± 42	Threonine	-0.98	-0.96	0.92	0.62	0.80
Histidine	700 ± 73	89	260 ± 26	190 ± 30	159 ± 23	170 ± 15	134 ± 16	149 ± 19^	Histidine	-0.95	-0.93	0.96	0.75	0.88
Valine	433 ± 36	252	257 ± 48*	208 ± 42	178 ± 39	187 ± 31	139 ± 29	152 ± 20	Valine	-0.95	-0.93	0.90	0.62	0.78
Glutamic acid	393 ± 47	60	168 ± 31*	140 ± 27	122 ± 21	121 ± 12	95 ± 11	106 ± 14	Glutamic acid	-0.94	-0.92	0.91	0.60	0.78
Leucine	335 ± 35	160	162 ± 18*	118 ± 13	102 ± 13	103 ± 8	95 ± 8	95 ± 8^	Leucine	-0.95	-0.96	0.99	0.80	0.93
Proline	252 ± 33	239	153 ± 30	114 ± 24	101 ± 22	106 ± 17	87 ± 14	89 ± 13	Proline	-0.97	-0.96	0.96	0.74	0.88
Tyrosine	292 ± 33	72	130 ± 20*	93 ± 15	78 ± 14	77 ± 9	69 ± 8	69 ± 7^	Tyrosine	-0.96	-0.96	0.98	0.76	0.91
Isoleucine	224 ± 22	84	129 ± 19*	94 ± 15	81 ± 14	83 ± 11	57 ± 12	67 ± 11^	Isoleucine	-0.95	-0.92	0.93	0.67	0.83
Lysine	265 ± 38	198	110 ± 20*	82 ± 14	66 ± 13	67 ± 10	53 ± 8	65 ± 8^	Lysine	-0.91	-0.90	0.96	0.74	0.88
Phenylalanine	221 ± 25	65	95 ± 11*	67 ± 8	56 ± 8	57 ± 5	52 ± 5	53 ± 5^	Phenylalanine	-0.94	-0.95	0.99	0.81	0.93
Hydroxy proline	93 ± 81		82 ± 63	62 ± 50	47 ± 40	41 ± 33	8 ± 6	30 ± 29	Hydroxy proline	-0.87	-0.82	0.84	0.49	0.69
Asparagine	144 ± 15	49	79 ± 20*	60 ± 14	47 ± 13	47 ± 13	40 ± 13	43 ± 8	Asparagine	-0.94	-0.94	0.96	0.70	0.86
Tryptophan	87 ± 15	30	38 ± 7*	24 ± 6	18 ± 6	17 ± 4	13 ± 4	13 ± 3^	Tryptophan	-0.97	-0.96	0.98	0.74	0.90
Glutamine	4 ± 4	645	17 ± 9	13 ± 9	12 ± 9	5 ± 5	10 ± 6	9 ± 7	Glutamine	-0.73	-0.74	0.83	0.50	0.72
Methionine	43 ± 7	32	11 ± 3*	6 ± 2	4 ± 3	4 ± 2	4 ± 3	6 ± 2^	Methionine	-0.76	-0.79	0.92	0.88	0.92
Hydroxylysine	8 ± 7		8 ± 6	9 ± 8	5 ± 5	6 ± 6	3 ± 3	2 ± 2	Hydroxylysine	-0.82	-0.81	0.62	0.16	0.40
α-aminoadipic acid	14 ± 6		6 ± 4	5 ± 4	5 ± 5	2 ± 2	2 ± 2		α-aminoadipic acid	-0.88	-0.86	0.78	0.33	0.61
α-aminobutyric acid	5 ± 5		5 ± 4	22 ± 19					α-butyric acid	-0.33	-0.39	0.23	-0.23	-0.03
Total	11,584 ± 831	2,962	6,422 ± 1187*	4,975 ± 951	4,231 ± 870	4,305 ± 632	3,534 ± 541	3,639 ± 497	Total	-0.97	-0.96	0.95	0.69	0.85

The significant differences between the passive sweat and the sweat collected after 15 minutes of exercise have been marked at p<0.05 (*). Significant differences during the exercise period are marked ^ (p<0.05). Significant correlations between the mean values of Na+, Cl-, K+, Mg2+ and Ca2+ and the twenty-three most abundant amino acids over the six exercise time points have been marked in italics (p<0.05).

The first sweat samples taken after 15 minutes of exercise showed marked differences in composition to the passive sweat samples (Table 1). The mean [Na+] and [Cl-] were increased by 8.9 mM and 6.25 mM respectively after 15 minutes of exercise compared with passive sweat, although these changes were not statistically significant. The [K+] was reduced by 2.1 mM and there were significant reductions in [Mg2+] and [Ca2+] of 0.08 and 0.29 mM respectively (p<0.05). There was a significant reduction in the average total level of amino acids compared to the passive sweat (6.4 vs 11.6 mM) (p<0.05). Individual amino acids serine, glycine, ornithine, valine, glutamic acid, leucine, tyrosine, isoleucine, lysine, phenylalanine, asparagine, tryptophan and methionine were significantly lower in the exercise sweat at 15 minutes than in the passive sweat samples (p<0.05, Table 1).

Electrolyte concentrations changed over the duration of the exercise period. The [Na+] and [Cl-] increased from 23.3mM and 17.2mM at 15 minutes (p<0.05), to 34.6 mM and 28.6 mM respectively at 65 minutes (Fig 1(A)). In contrast, the total amino acid concentrations in sweat reduced from 6.4 mM at 15 minutes to 3.6 mM at 65 minutes although this was not significant. The [K+] in exercise sweat declined from 8.2mM to 5.5mM at 65 minutes, but the [Mg2+] remained relatively constant throughout the period of exercise (Fig 1(B)). [Ca2+] decreased between 15 and 25 minutes but remained stable for the remaining 50 minutes of exercise.

Fig 1

(a) Concentrations of Na+ and Cl- (primary axis) and mean total amino acid concentrations (secondary axis) in exercise sweat (mM) assessed at ten minute intervals from 15 to 65 minutes of exercise (n = 11). The concentrations of Na+, Cl- and total amino acids in passive sweat (n = 6) are also shown for reference on the respective axes. (b), Mg2+ and Ca2+ concentrations (primary axis) in exercise sweat for the exercise duration are shown with amino acid and K+ concentrations (secondary axis).

As predicted, the [Na+] and [Cl-] in the exercise sweat were positively correlated throughout the exercise period (r = 0.99, p<0.05, Table 1). Neither [Na+] nor [Cl-] were positively associated with any other electrolyte or amino acid in the sweat but showed significant negative correlations with the total amino acid concentrations (r = -0.97, r = -0.96, p<0.05), K+ (r = -0.93,p<0.05) and Ca2+ (r = -0.83, p<0.05). Significant negative associations were demonstrated between [Na+] and twenty of the individual amino acids measured, while [Cl-] was negatively correlated with nineteen of the twenty three amino acids assessed during the exercise period (Table 1). The [K+], [Mg2+], [Ca2+] in sweat were all positively correlated with each other (r > 0.82, p<0.05). [K+] was positively correlated with twenty of the amino acids (p<0.05), [Ca2+] was correlated with fifteen amino acids (p<0.05) and [Mg2+] was associated with three amino acids (p<0.05).

Discussion

The current study aimed to investigate potential relationships between [Na+] and [Cl-] with [K+], [Mg2+], [Ca2+] and the amino acids in passive and exercise sweat collected from recreationally active participants over a defined period of cycling exercise. The results indicated that passive sweat collected over a 15 minute period had higher [K+], [Mg2+] and [Ca2+] and concentrations of amino acids compared to sweat collected after 15 minutes of exercise. In contrast, the [Na+] and [Cl-] in the passive sweat were lower than those observed in the 15 minute exercise sweat. These differential profiles of amino acids and electrolytes in the passive and exercise sweats suggested that the composition of sweat was physiologically regulated by exercise. The advent of exercise would require the body to maintain sweating capacity to counter the higher heat generation for energy consumption in the muscles for prolonged periods and so a more conservative approach for retention of Na+ and Cl- would be required. Under these conditions of exercise, the body would limit the rate of losses of the amino acids and K+, Ca2+ and Mg2+ and, as a result, the capacity to resorb the Na+ and Cl- would be reduced, resulting in higher [Na+] and [Cl-] in exercise sweat. It should be noted that K+ was the only ion in the exercise sweat at a concentration (8.2mM) greater than the literature average value in plasma (4.4mM). This would be consistent with K+ having a major role in the process of sweat formation and/or resorption of Na+.

During the exercise period, it was observed that there were progressive increases in in [Na+] and [Cl-] in the sweat, whilst the [K+], [Mg2+] and [Ca2+] and amino acids diminished. In the current study, the exercise intensity and environmental conditions were kept constant to enable a constant sweat rate [6, 30]. The progressive increases in [Na+] and [Cl-] were thus interpreted to indicate that the capacity to resorb Na+ and Cl- during passage through the duct diminished over the exercise period. In contrast, the [K+] in the sweat fell after 15 minutes of exercise and continued to decrease over time to levels that were closer to, but still higher than, the literature plasma levels by 65 minutes. The mean total level of amino acids was also approximately twice the literature plasma value after 15 minutes exercise and progressively diminished over time approaching levels equivalent to plasma by 55–65 minutes. As a result, there were strong positive correlations between the sweat concentrations of amino acids and [K+], with both these components having strong negative correlations with [Na+] and [Cl-]. These results suggested inverse physiological relationships between [Na+] and [Cl-] in the sweat with [K+] and the amino acids which were modulated by the onset and duration of exercise.

The in vivo amino acid content of the primary sweat fluid produced in the secretory coil has not been examined independently of sweat leaving the resorptive duct, as was achieved with in vitro studies of sweat electrolytes from excised glands [31]. It would be reasonable to assume that the amino acid levels in the primary sweat fluid formed in the secretory coil would be less than or similar to plasma levels with no published literature evidence of amino acid transporters operating in this part of the gland or throughout the transdermal duct. A potential transporter that facilitates the exchange of amino acids for extracellular substrates is the ASCT-1 protein of the alanine/serine/cysteine transporter family (ASCT) [32]. The expression of ASCT-1 mRNA was found to be expressed in all the human tissue types that were tested by northern blot, with the highest abundances occurring in the brain, muscle and pancreatic tissue [33]. The ASCT-1 protein transports alanine, serine, cysteine and threonine in a symmetrical and electroneutral exchange of one of these zwitterionic amino acids with one Na+ ion [34]. As the ASCT-1 has a high affinity for serine and alanine, the presence of this transporter in the duct of the sweat gland may provide a supplementary mechanism by which Na+ is reclaimed at the cost of small, neutral amino acids. The operation of this transporter would be consistent with the very high levels of serine and alanine measured in the sweat. Other amino acid transport systems are likely to be involved given the strong inverse correlations between [Na+] and [Cl-] with the concentrations of eighteen amino acids, [K+], [Mg2+] and [Ca2+] in the sweat.

Once the primary sweat fluid has been formed in the secretory coil, the amino acids and K+ could be transferred to the primary sweat fluid during passage through the intradermal duct to balance the resorption of Na+ and Cl-. It has been proposed that K+ is lost from the duct cells via leak channels as a consequence of Na+ resorption in an attempt to maintain osmotic balance in the cells but not necessarily as a direct part of the Na+ transport channel operation [18, 31]. In this context, Na+ would be resorbed from the primary sweat fluid into the cells via the ENaC channels as it travels along the duct. The change in the ion concentration gradient causes K+ to exit the cell passively through K+ leakage channels, resulting in elevated K+ concentrations in the sweat at the skin surface. The amino acids may be linked to this process or have independent mechanisms of transfer. Ca2+ and Mg2+were also implicated in the resorption process by showing positive correlations with K+ and the amino acids. Whilst Ca2+ has been linked to KCl efflux channels [24, 35, 36], the full role and mode of action of Mg2+and Ca2+ has not been clearly elucidated and the connection between the cations and amino acids is at present undefined.

The increases in [Na+] and [Cl-] throughout the exercise period indicated that the capacity for resorption diminished over this period. It was thus proposed that whilst sufficient amino acids and K+ were available, the Na+ can be effectively resorbed but as exercise and sweating continues and reserves of amino acids and K+ diminish, then Na+ (and Cl-) would not be as effectively resorbed and thus Na+ and Cl- would begin to increase in concentration within the sweat matrix. If the concentrations of amino acids and K+ became limiting within the duct cells with exercise duration, then fewer Na+ ions could be resorbed progressively resulting in higher Na+ and lower K+ concentrations in sweat as was observed in the present study.

Conclusions

The data from this study supported the hypothesis that key amino acids in combination with K+, Mg2+ and Ca2+, are transferred into the sweat to facilitate resorption of Na+ and Cl-. The involvement of the amino acids, K+, Mg2+ and Ca2+ in the resorption of Na+ and Cl- would provide an explanation for the high losses of these components via sweat during exertion. During physical activity, reclamation of Na+ and Cl- is important in avoiding exercise-associated hyponatremia (EAH). The implication for professional and recreational athletes is that provision of electrolytes alone would not be sufficient to balance the replenishment requirements generated by prolonged exertion. These results could provide new insights in sports medicine to manage athletes undertaking high intensity and endurance training or competition, especially in warmer climates. It would even be possible to use measures of sweat-facilitated losses of amino acids to customise supplementation to minimize the supply from endogenous turnover of body proteins. Ideally, the amino acids and electrolytes would be replaced in a similar concentration profile as to that in which they are lost, if possible during, or immediately after exercise.

12 Sep 2019

PONE-D-19-20805

Relationships between electrolyte and amino acid compositions in sweat during exercise suggest a role for amino acids and K+ in reabsorption of Na+ and Cl- from sweat

PLOS ONE

Dear Dr R. Hugh Dunstan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR:

I suggest to

a) include all the analytical figures of merit of the procedures used to analyse sweat samples

b) conclusions, underline the possible applications of these results in the field of the sport medicine.

c) introduction, explain the reasons of selecting sweat samples instead of other non-conventional biological fluids (e.g. saliva). Saliva analysis is a well-know approach to obtain information of the physiological status in both clinical and sport medicine field. As example, the following articles can be useful for the authors and used in the introduction.

DOI: 10.1016/j.microc.2017.04.033

DOI: 10.1016/j.microc.2017.02.010

DOI: 10.1016/j.aca.2017.07.050

Sports Med. 1998 Jul;26(1):17-27.

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Academic Editor

PLOS ONE

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this work the authors' purpose was to evaluate the potential relationships between [Na+] and [Cl-] with [K+], [Mg2+], [Ca2+] and the amino acids in sweat, and the role of exercise on sweat composition. The authors showed that the exercise stimulates a change in sweat composition with an increase of [Na+] and decrease of amino acids concentration. The results suggest that amino acids, [K+], [Ca2+] are associated with reabsorption of [Na+] and [Cl-].

The work turns out to be well written, the sources cited are appropriate.

The idea turns out to be innovative and the results are very interesting. Indeed, the results will lay important bases on understanding biochemistry of physical exercise and physiological interactions. Not only that, they will also allow to better understand the type of supplement to be carried out during a training and if it is the case to carry it out.

I have only few questions:

a) The authors did not carry out the assessment of body composition in terms of fat and lean mass.

Do you think that there could be an influence in the results in relation to the change in fat and lean mass percentages?

b) The exercise in the study is of an aerobic nature.

According to by Kerksick et al. (2018) [Kerksick CM, Wilborn CD, Roberts MD, et al. ISSN exercise & sports nutrition review update: research & recommendations. J Int Soc Sports Nutr. 2018 Aug 1;15(1):38. doi: 10.1186/s12970-018-0242-y], the position of the International Society of Sports Nutrition (ISSN) in order to achieve an improvement in performance, the only amino acid (AA) that seems to play a role is beta-alanine, while for the other AA the results they are contradictory.

Because on their results, do the authors believe that an integration with essential amino acids (EAAs) and branched amino acids (BCAAs) could improve performance? If yes, when would it be fairer to hire them? Before, during or after the performance?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Sep 2019

This is more readily viewed in the response to reviewers file submitted as an attachment.

Response to Reviewers:

ACADEMIC EDITOR:

I suggest to

a) include all the analytical figures of merit of the procedures used to analyse sweat samples

This method has been described previously in our earlier PLOS publication on human sweat. We should have referenced this before. This has been added in lines 128 – 9.

b) conclusions, underline the possible applications of these results in the field of the sport medicine.

The conclusions have been amended as requested.

c) introduction, explain the reasons of selecting sweat samples instead of other non-conventional biological fluids (e.g. saliva). Saliva analysis is a well-know approach to obtain information of the physiological status in both clinical and sport medicine field. As example, the following articles can be useful for the authors and used in the introduction.

DOI: 10.1016/j.microc.2017.04.033

DOI: 10.1016/j.microc.2017.02.010

DOI: 10.1016/j.aca.2017.07.050

Sports Med. 1998 Jul;26(1):17-27.

The intent of the study was not to try to determine physiological status of amino acids in the body. We specifically set out to investigate the nature of amino acid losses in sweat during exercise and how this might be related to losses of electrolytes. Sweat losses can occur at rates of 1-2 L per hour depending on the exercise intensity, type and external conditions. Thus losses of electrolytes and amino acids can be considerable.

In the introduction, it is pointed out that sweat contains certain amino acids at very high concentrations (line 44… and again at line 82) – many times higher than in plasma, but little is known as to why they are there at high levels – what function are they serving?

The perspective of sweat as a constructed fluid is then presented which is well understood in terms of electrolyte composition and re-absorption of Na and Cl, but there is nothing published about how and why the amino acids actually enter the sweat fluid.

The hypothesis is thus proposed that the amino acids and K may have direct roles in Na resorption. The project thus focusses on gathering data that might support this hypothesis and shed light on mechanisms.

We would appreciate receiving your revised manuscript by 29th September. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Tommaso Lomonaco, Ph.D

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

We have altered the referencing format as requested.

We have changed the headings style as requested.

We have changed the author naming as requested and amended the information for the corresponding author. We have also marked the “equally contributing” authors.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this work the authors' purpose was to evaluate the potential relationships between [Na+] and [Cl-] with [K+], [Mg2+], [Ca2+] and the amino acids in sweat, and the role of exercise on sweat composition. The authors showed that the exercise stimulates a change in sweat composition with an increase of [Na+] and decrease of amino acids concentration. The results suggest that amino acids, [K+], [Ca2+] are associated with reabsorption of [Na+] and [Cl-].

The work turns out to be well written, the sources cited are appropriate.

The idea turns out to be innovative and the results are very interesting. Indeed, the results will lay important bases on understanding biochemistry of physical exercise and physiological interactions. Not only that, they will also allow to better understand the type of supplement to be carried out during a training and if it is the case to carry it out.

I have only few questions:

a) The authors did not carry out the assessment of body composition in terms of fat and lean mass.

Do you think that there could be an influence in the results in relation to the change in fat and lean mass percentages?

This could be relevant. Eccrine glands do not have “fatty” secretions like the apocrine glands but there could be a link between the body types and sweat composition. Our first study indicated that concentrations of amino acids could vary between individuals, which may be related to various body types and fat composition. Future work could certainly target comparisons of sweat content with fat and lean mass.

b) The exercise in the study is of an aerobic nature.

According to by Kerksick et al. (2018) [Kerksick CM, Wilborn CD, Roberts MD, et al. ISSN exercise & sports nutrition review update: research & recommendations. J Int Soc Sports Nutr. 2018 Aug 1;15(1):38. doi: 10.1186/s12970-018-0242-y], the position of the International Society of Sports Nutrition (ISSN) in order to achieve an improvement in performance, the only amino acid (AA) that seems to play a role is beta-alanine, while for the other AA the results they are contradictory.

Because on their results, do the authors believe that an integration with essential amino acids (EAAs) and branched amino acids (BCAAs) could improve performance?

Our research has also focussed on modelling amino acid turnover with specific reference to including exact losses via excretory pathways of sweat and urine. This modelling shows that a select few amino acids are subject to short supply as exercise demand increases: either as essential amino acids or conditionally essential amino acids. During exercise, some amino acids are utilised at faster rates than others, and these are supplied endogenously by catabolism of muscle proteins. See our ref below:

Dunstan, R. H., Macdonald, M. M., Murphy, G. R., Thorn, B., & Roberts, T. K. (2019). Modelling of protein turnover provides insight for metabolic demands on those specific amino acids utilised at disproportionately faster rates than other amino acids. Amino Acids. doi:10.1007/s00726-019-02734-1

We did not reference this article in this manuscript because here, the focus was on the associations between amino acids and electrolytes during the course of exercise.

The modelling paper basically infers that if you can provide the amino acids that are lost at disproportionately faster rates than others, then you could reduce the need for endogenous turnover of proteins. This would minimise wastage of amino acids that are not necessarily required in high demand – such as the branch chain amino acids.

If yes, when would it be fairer to hire (=take) them?

It would be proposed to take an amino acid supplement that was designed to replenish those most rapidly lost during the exercise via sweat. Since digestion is impaired during exercise, it is possible to provide the free amino acids that can be directly absorbed to replenish those lost at the faster rates.

Before, during or after the performance?

All three stages are possible. It should be noted that digestion can be impaired during and for several hours after the exercise, dependent on the intensity and duration. However, the repair and recovery processes are underway and continue after cessation of exercise, even if digestion is minimal. So it would be recommended to take amino acid supplementation as soon as possible after exercise. It is also proposed that taking a combination mix of electrolytes and amino acids during exercise would be advantageous.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Submitted filename: Response to Reviewers 16092019.docx

Click here for additional data file.

20 Sep 2019

Relationships between electrolyte and amino acid compositions in sweat during exercise suggest a role for amino acids and K+ in reabsorption of Na+ and Cl- from sweat

PONE-D-19-20805R1

Dear Dr. Dunstan,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Tommaso Lomonaco, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Authors,

the present version of the article is fine and can be accepted in PlosOne for publication.

Best regards,

Tommaso Lomonaco

Reviewers' comments: