Materials and Apparatus

Bioengineering 210

Laboratory II

Final Project: Analysis of Ionic Concentration of Sweat Using the Atomic Absorption Spectrophotometer

Professor Dr. Mitchell Litt

SPRING 1998 Group W6 Paul Kim Catherine LaRocco Dale Yoo Hua Zhu ABSTRACT

In this experiment, three sweat collecting methods and devices were designed and tested. Among the three methods and devices, a simple sweat-catching method using test tubes and a slightly more complicated method using a flexible pouch constructed with Opsite-type dressing and parafilm were tested and determined to be inaccurate or inefficient because of various factors. The third device was constructed with a 100 mL square petri dish and syringe. It was tested under experimental conditions and proved to be the most efficient for easy sweat collection. It was used to collect all of the sweat samples in this experiment because of its ability to prevent contamination of the sweat sample, thus, ensuring the accuracy of the measurements and data. In the second part of the experiment, sweat samples were collected in dry and steam sauna conditions with mean temperatures of 185 F and 156 F, respectively. The relative humidity was negligible in the dry sauna and 100 % in the steam sauna. The ion concentration of Na+, K+ and Ca2+ in the sweat samples were determined using an atomic absorption spectrophotometer. According to previous research conducted regarding sweat content, the first data points of the potassium and calcium concentration do not truly reflect the concentration of ions in the sweat. This was supported by our data. Additionally, we found no obvious effect of relative humidity on ion concentration in sweat except for Ca2+ ion concentration, where the dry trials yielded much higher calcium concentrations than in the steam sauna. Since there was no distinction between the physiological conditions overall, we calculated the mean ion concentrations of all trials. The mean concentration of Na+, K+ and Ca2+ in sweat was found to be 1884 ppm, 236.0 ppm, and 8.441 ppm, respectively. The experimental sodium ion concentration was lower than the normal blood ion concentration as well as literature values for drug- induced sweat production but was higher than the literature value for thermally induced sweat. On the other hand, the experimental potassium ion concentration was higher than the normal blood ion concentration and the drug-induced sweat but lower than the thermally induced sweat. Calcium ion could not be compared to literature values because of the lack of data for this ion.

2 BACKGROUND

There are many forms of sweating that occur in the human body: eccrine, emotional, gustatory, and apocrine, although eccrine is the only form to secrete a significant amount of fluid and electrolytes. The other forms occur sporadically and often have inconsequential fluid loss. Under stress, a man is capable of sweating at rates as high as 2 liters/hr and total volumes in excess of one quarter their total body fluid in a single day.[1] The secretion of sweat glands, a type of exocrine gland, cools the body by evaporation and, at least in lower life forms, produces a sexual attractant odor.[2] The millions of eccrine glands produce an odorless, light and watery[3] diluted salt solution for thermoregulation,[4] while the bacteria around the apocrine glands produce odor-causing byproducts as waste.[3] These bacteria are nourished from the glands’ protein-rich secretion.[4] Deodorants are antimicrobial agents that minimize the number of bacteria and fragrances to mask any remaining undesirable smells. Antiperspirants are aluminum salts such as the very potent and irritating aluminum chloride or the least powerful and least irritating aluminum chlorohydrate, which plug up sweat gland ducts to stop odor and decrease the amount of wetness.[3] The literature shows that pharmacologically induced sweat contains 3381 ± 253 ppm of sodium and 195.5 ± 39.1 ppm of potassium.[5] (There are no values for the concentration of calcium in sweat because it is found in trace amounts.) Various researchers agree that the range of ion content of sweat falls within or close to the range of ion content of blood (Table 1).[5,6]

Sodium Reference Range 3128 ppm – 3358 ppm Calcium Reference Range 48.50 ppm – 52.91 ppm Potassium Reference Range 136.85 ppm – 195.5 ppm Table 1: Ion Content of Blood[6]

3 It has been found, though, that the human sweat duct utilizes an ATP-dependent pump (Figure 1). The research of Sato, Fiebleman, and Dobson indicates that this pump can cause lower sodium and higher potassium concentrations than expected from pharmacological data. [7] This type of pump, or primary active transport carrier,[8]

Figure 1: Classic Na+/K+ uses the energy derived from ATP to drive the Electrogenic Pump[9] simultaneous active transport of Na+ ions out of and K+ ions into the cell,[9] both against their respective concentration gradients. One cycle of the constantly active pump [8] moves three Na+ ions and two K+ ions. This pump exists in animal cells and uses a large percentage of the metabolic energy of the cells.[9] It is what keeps the constant[10] low [Na+] and high [K+] in the cytoplasm.[9] In this study of sweat, it was determined that an analysis of a sports drink would help one to understand thermally induced sweat. As a case study, Gatorade Thirst Quencher®, a registered trademark of Stokely-Van Camp, Inc., was investigated. According to this company, a sports drink should do all of the following: stimulate rapid fluid absorption, assure rapid rehydration, provide carbohydrate energy to working muscle, and encourage more drinking of fluids.[11] Gatorade Thirst Quencher® contains 0.4583 g/L (458.3 ppm) of sodium and 0.1250 g/L (125.0 ppm) of Figure 2: Gatorade Label [12] potassium.[12] Stokely-Van Camp, Inc.’s research has shown that beverages containing about a 6 % carbohydrate level and a small amount of sodium are ideal for rapid fluid absorption. But as the carbohydrate percentage of a drink is increased, absorption is slowed. For this reason, drinks that contain carbohydrate levels that are greater than 7 % are not recommended. Complete restoration of body fluids is best when the sodium that was lost in sweat is replaced along with fluids. In addition, the sodium and glucose levels in

4 Gatorade stimulates people to drink more fluid voluntarily until the body is rehydrated.

Sweat is a biological fluid that is often overlooked, so there still remains many technical problems such as dilution, condensation, contamination, and evaporation, that must be solved when developing a sweat collection device. Brisson et al. have developed a technique using an OpSite-like medical dressing (Figure 3). Before application on a cleaned and dried surface of the mid-lumbar region of the subject’s skin, the adhesive side of the dressing is exposed and a Figure 3: Design 2 Schematic of Sweat Collector using Smith & Nephew's OpSite Wound piece of permeable laboratory film paper, such as Dressing[14] parafilm, is attached. The dressing is then transferred to the skin forming a “pocket”. A small opening is created in the upper part, but it is kept closed when the sweat is not being extracted.[14] The sweat can be suctioned from the dressing by using a Vacutainer® tube inserted in a tube holder and fitted with a long dull needle.[15] Boisvert et al. modified the previously mentioned device so that it would allow collection in a warm (30 C) and humid (relative humidity 80 %) environment. This new technique would reduce leakage and hidromeiosis during intense or prolonged exercise. The first modification was the application of hypo-allergic glue, which is made of benzion simple tincture and binds the OpSite-like membrane to the surface of the skin. This has been shown to prevent leaks when collection extends over one hour or is performed in humid environments. Secondly, the excreted sweat was diverted to a pouch chamber attached to the lower end of the device. This permitted the sweat to accumulate away from the skin to minimize potential hidromeiosis and changes in skin temperature. [15] Another method, by Taylor, Polliack, and Bader, utilized a 4 cm  4 cm square of Whatman cellulose chromatography paper for sweat collection. This paper is covered with a water-impermeable sheet of 50 m thick polypropylene and sealed around the edges with surgical tape. After collection, the cellulose pad was placed in a tube and

5 deionized water was added. The tubes were centrifuged at 3000 G for 10 minutes in order for the sweat to accumulate.[16]

MATERIALS and APPARATUS

Preparation of Calibration Solutions  Solutions of 1000 ppm K+, Ca+2, and Na+  Fisherbrand® plastic 15-mL and 50-mL disposable test tubes and Nalgene® test tube racks  4 plastic 100-mL disposable beakers  2 plastic 1000-mL disposable beakers  Denville XL 3000 20-200 L and 100-1000 L pipettes  Fisherbrand® Redi-Tip 200-L and 1000-L pipette tips  Drummond Scientific Co. pipet-aid®  Fisherbrand® disposable 10-mL pipettes  Scoopula  Deionized water

Sweat Collection  Becton Dickinson Vacutainer® Blood Collection Set  Puncture needle  Long dull needle  Johnson & Johnson Medical Inc. Bioclusive* transparent dressing  Paraflim  Fisherbrand square petri dish with grid (13-mm  33-mm  33-mm)  Tycon tubing (1/32 inch od - 3/32 inch od, 1/16 inch id - 1/8 inch od, 1/8 inch id - 1/4 inch od)  Super glue  Cyclohexanone  20-mL Becton Dickinson syringe  3-foot  10-inch (approximate) latex Thera-Band® strip  10-mL test tubes  Kendall Webcol® Alcohol Prep, Sterile, Saturated with 70 % Isopropyl Alcohol  Paper towel  Deionized water

Determination of Ion Content  Perkin-Elmer Atomic Absorption (AA) Spectrophotometer 4000  Microsoft Excel®  Deionized water

6 METHODS

There were two parts in this experiment: 1) to design an efficient, reliable and reusable sweat collecting device and 2) to determine the concentration of sodium, potassium, and calcium ions in sweat.

Design 1 Our first method, which was the most primitive one, was to swab a 10-mL test tube against the subject’s skin to collect the sweat.

Design 2 The second method was a simple disposable device designed after studying several scientifically related research papers and consulting Dr. Henry R. Drott, an expert in the field of cystic fibrosis and sweat collection at the Children’s Hospital of Philadelphia (see Acknowledgements). This device was designed to apply the special properties of OpSite-type medical dressing. It was constructed with a piece of parafilm cut into the shape that would collect sweat at the bottom of the device when applied vertically on the lumbar region of the subject’s back (Figure 3). It was fixed by applying a piece of OpSite-type medical dressing covering the entire device, thus, creating a small airtight pouch under the parafilm for collecting sweat. Then, the sweat collected at the bottom of the pouch would be extracted with a needle connected to a Vacutainer®, which sucks the sweat from the pouch into the tube. Because of the special properties of the dressing, it would adhere to the skin firmly even under humid conditions, such as those created by sweating.

Design 3 After receiving advice from Gia Hchevia, a biotechnologist and researcher at Hospital of the University of Pennsylvania (see Acknowledgements), a third device was designed to solve the problems encountered in the first two. This device was made of a square petri dish of 13-mm  33-mm  33-mm. A size 1/32 inch inner diameter - 3/32 inch outer diameter tube was glued to a hole (diameter 2/32 inch) drilled at one of the

7 corners of the petri dish. At the other end of the tube, a 20-mL syringe was attached for collection (Figure 4). The device was applied to the lumbar region of the subject’s back and fixed with a wide latex strip (Figure 5). An airtight space was created between the petri dish and the subject’s skin, and the sweat was extracted from the plastic tube by a syringe.

Sweat Collection Using the third design, which was determined to be the most efficient and accurate, sweat samples of the same subject from four trials under two distinct conditions were collected Figure 4: Design 3 Sweat Collection Apparatus and analyzed. Two trials were carried out for each of the two environmental conditions, which were created by a steam sauna and a dry sauna. In the dry sauna, the temperature was approximately 185 F with negligible humidity. In contrast, the temperature in the steam sauna was 156 F with relative humidity of 100 % since the air was saturated with water vapor. A sweat sample was collected every 15 minutes in each trial and the subject was allowed to rest for 5 minutes during extraction of the sample to prevent severe dehydration. Between each trial, the subject rested 1 hour and drank 1 liter of deionized water to recover to his normal state.

Preparation of Calibration Curves To analyze the ion content of sweat using an atomic absorption spectrophotometer, calibration curves must first be constructed for Figure 5: Application of Design 3 onto each ion that will be tested before each run with Lumbar Region of Subject the machine. The 1000 ppm stock solutions of potassium ions (K+), calcium ions (Ca+2), and sodium ions (Na+) must be diluted in order to find the linear region of the absorbance

Figure 6: Typical Absorbance versus Ionic Concentration Calibration Curve versus concentration curve. Using the method of parallel dilutions, these solutions were diluted to 100 ppm, 50 ppm, 10 ppm, 8 ppm, 6 ppm, 4 ppm, and 2 ppm, making 50 mL of each. The absorbance of each of these solutions was determined three different times and then the averages of these three determinations were plotted as a function of their respective concentrations. A best-fit line was formed by linear regression and its equation was calculated using the Microsoft Excel® computer program. By plotting such low concentrations, we would obtain an accurate linear region for the calibration curves, which typically look like Figure 6.

Determination of Ion Content Before actually measuring any values from the sweat, we diluted the sweat samples for two reasons. First, we only had approximately 1 mL of Absorbance the each sweat sample so if we ran the sample through the AA spectrophotometer without diluting it, Concentration we would have lost all of our sample in that one measurement. Secondly, the sweat Figure 6: Typical Absorbance versus Ionic Concentration Calibration Curve sample was too concentrated to fall into the linear range of our calibration curves. At the start of our experimentation, we had literature values from Bijman[5] to predict what the concentrations of the sweat samples might be. So we decided to dilute the sample by different dilution factors, which were calculated specifically for each ion. We initially decided to dilute all the samples from both of the dry sauna trials by a factor of 50. We found that at this dilution factor, the Na+ concentrations in the samples were still above the linear region of the calibration curve ([Na+] >10 ppm). The K+ concentrations fell consistently within the linear range (2 ppm < [K+] < 10 ppm) while the Ca+2 concentrations were below the linear range ([Ca+2] < 2 ppm). Therefore, we decided to further dilute the samples to a dilution factor of 1000, and we observed that the [Na+] readings finally fell within the linear region of the calibration curve for Na+. We

9 determined that it was acceptable for the calcium ion to have concentrations below 2 ppm because we assumed that the amount of absorption was zero at zero concentration. Hence, we could extrapolate the best-fit line by adding the point (0,0) to our regression line. [Ca+2] of the sweat samples would now fall into the linear region of the calibration curves. So for the wet sauna samples collected the week after, we diluted each sample by a factor of 10, 50, and 1000 to test for the [Ca+2], [K+], and [Na+], respectively. Note that we changed the dilution factor of the calcium ion from 50 to 10 so that the data point would be closer to the range that we actually investigated (2 ppm < X < 10 ppm). The [Ca+2] values were still lower than 2 ppm at a dilution factor of 10; however, we could not use a lower dilution factor because we would not have enough of the sweat sample to obtain all the appropriate absorption readings from the AA spectrophotometer. For each sweat sample, we took three readings and averaged them. We used these averages to determine their respective concentrations by using the calibration curves.

10 RESULTS

The following graphs represent all of the collected data for each of the three ions after using the appropriate calculations:

Concentration versus Time for Na+

3000 ) m p

p 2500 (

a 2000 Dry Sauna Trial 1 N

o Dry Sauna Trial 2

n 1500

o Steam Sauna Trial 1 i t a

r 1000 Steam Sauna Trial 2 t n e c 500 n o C 0 0 20 40 60 80 Time (min)

Figure 7: Calculated Concentration of Sodium Ions versus Duration of Experiment - The four trials are represented by theFigure different 8: data Calculated point patterns, Concentration as indicated of in Calcium the legend. Ions versus Duration of Experiment - The four trials are represented by the different data point patterns, as indicated in the legend.

Concentration versus Time for Ca+2

) 100.0 m p p

( Dry Sauna Trial 1

2 80.0 Dry Sauna Trial 2 a C

f 60.0 Steam Sauna Trial 1 o

o Steam Sauna Trial 2 i

t 40.0 a r t n

e 20.0 c n o

C 0.0 0 20 40 60 80 Tim e (m in)

11 Consistency of Trial 2 and Trial 1

) 250 + m Concentration versus Time for K p p ( 200

n y = x o

a R = 1 r )

t 150 m n 500 p e p c (

n + 100 Dry Sauna Trial 1

o 400 K C

f Dry Sauna Trial 2 2 o

n 300 a o i i Steam Sauna Trial 1 r The t a T r 0 t 200 Steam Sauna Trial 2 n e following c 0 50 100 150 200 250 300 table n

o 100 C Trial 1 Concentration (ppm) represents 0 the 0 20 40 60 80 results of t- tests Tim e (m in) that were run to compare the two separate trials within the same condition for each ion. Figure 9: Calculated Concentration of Potassium Ions versus Duration of Experiment - The four trials are represented by the different data point patterns, as indicated in the legend. Type of Trial Comparison Mean - Trial 1 Mean - Trial 2 t Stat t Critical two-tail [Na+] in Dry 1596.89 2249.06 -1.997 2.447 [Na+] in Steam 1735.83 1952.81 -2.195 2.447 [K+] in Dry 155.35 245.03 -2.229 2.447 [K+] in Steam 178.81 208.69 -1.118 2.447 [Ca+2] in Dry 35.07 19.68 4.258 2.447 [Ca+2] in Steam 5.38 6.81 -1.123 2.447

Table 2: T-Test Results - This table shows the comparison of means between the two trials in each of the two conditions (dry sauna and steam sauna) for each of the three ion concentrations ([Na +], [K+], and [Ca+2]). Also, their respective t Stat and t Critical two-tail values are shown.

12 Figure 10: Ideal Graph of Trial 1 Concentration Data versus Trial 2 Data – This figure shows the ideal consistency between two trials. Since both trials are performed under the same condition (i.e. steam sauna or dry sauna), it is assumed that the ion concentration as a function of time is the same for both trials. Hence, in an ideal situation, the ion concentration at a particular time is the same for both trials since the external conditions have the same effect on both trials. It is assumed that no other variables exist that affect ion concentration between trials Ion Mean Sweat Mean Blood Ion Percent Concentration (ppm) Concentration (ppm) Deviation (%) Sodium 1884 3243 -41.9 Steam Sauna Trend Comparison for [Ca+2] Potassium 236.0 166.2 42.0 Calcium 8.441 50.71 -83.4 ) 25 m

p R2 = 0.961 p (

n 20 o i t a r

t 15 n e c

n 10 o C

l 5 a i r T 0 0 5 10 15 20 Trial 1 Concentration (ppm)

Figure 11: Graph of Trial 2 [Ca+2] versus Trial 1 [Ca+2] – This figure shows that there is consistency between the Trial 1 and Trial 2 data for calcium ion concentration in the sweat samples that were collected in the steam sauna. The R2 value of 0.961 shows that there is a strong correlation between the two sets of data.

13 Table 3: Mean Sweat Concentrations Compared to Blood and Reference Sweat Concentrations of Ions – Sodium, potassium, and calcium mean ion concentrations in our experiment in comparison to blood ion concentrations.[6]

Ion Experimental Drug Induced Thermal Induced Sweat Conc. (ppm) Sweat Conc. (ppm) Sweat Conc. (ppm) Sodium 1884 3381 648.0 Potassium 236.0 195.5 246.0 Calcium 8.441 ------

Table 4: Pharmacological versus Thermally Induced Sweat

14 DISCUSSION

Our experiment investigated the ion content of sodium, potassium, and calcium in sweat. Since the analysis of sweat entailed the actual sampling of sweat, the first aspect of our experiment was to design a device for collection. We tested three devices as shown in the Methods section. Device 1 simply involved the catching of sweat droplets as they fell from the subject’s forehead. Very quickly, this method was eliminated for several reasons. First, the catching of sweat into a test tube proved to be too tedious and complicated. We thought that we could just collect the sweat into a larger tube or funnel but that proved to be impossible because the sweat droplets adhered to the sides of the container and the funnel when they were transferred into smaller tubes. Additionally, this method would not yield accurate data because the water in the sweat had time to evaporate as the drops were collected. In addition, in trying to swab the sweat into the tubes, some epithelial cells were inevitably collected along with the sweat sample. These cells would need to be centrifuged out of the sweat. Otherwise, these cells would ultimately clog the aspirator of the AA spectrophotometer. Furthermore, this method was an “open” collecting design, which means that the collected sweat was in direct contact with the air and may have become contaminated. Boisvert[15] et al. previously reported that there was a great discrepancy of concentration data between sweat collected from an open system and a closed system. Therefore, some type of closed collecting system needed to be devised. The second device that was created was an imitation of a sweat collecting device described in an article by Brisson et al.[14] It consisted of a flexible “pouch” attached to the body that would be essentially airtight and prevent evaporation. Since evaporation would increase the measured concentrations of ions, measurements of samples from an open system would not be purely representative of sweat secreted by the body. Hence, this device should yield better results than the open system. However, this device had several problems. The OpSite-type dressing used to fix the device onto the subject’s body lost its adhesiveness when the skin under the dressing began to excrete sweat causing leakage from the pouch and making the collection of a sufficient amount of sweat within the designated time impossible. Furthermore, the sweat that was collected was too

15 concentrated since much of the water had evaporated from the sample from the leak in the pouch. The third device that was used was determined to be the most efficient and accurate device. It eliminated possible evaporation, minimized leakage, and ensured that all the sweat collected was from the designated region. Evaporation was obviously minimized because of the rigidity of the container as well as the airtight withdrawal system, which involved the use of a syringe. As the plunger was pulled, negative pressure was created within the petri dish, hence, maximizing the collection of sweat at the hole. Additionally, the specific location of the petri dish in the middle lumbar region was helpful because the skin surrounding the dish was flexible; this region was recommended by Brisson et al.[14] and Dr. Drott (see Acknowledgements). The second half of our experiment dealt with the actual measurement of sodium, calcium, and potassium ions in sweat during two different physiological conditions and comparisons of the results. The first physiological condition was a dry sauna with an average temperature of 185 F and negligible relative humidity. The second condition was a steam sauna with an average temperature of 156 F and relative humidity of 100 % since the air was fully saturated with water vapor. When analyzing the sweat samples that were collected under these two conditions, we tried to determine if there was any correlation between the sweat ion concentration and the humidity of the environment, in addition to the relationship between concentration and time. We predicted that the ion concentration of the sweat would be higher in the steam sauna than in the dry sauna. Our predictions were based on the fact that the human body tends to excrete higher concentrations of ions with increases in the sweat rate as previously documented by Boisvert[15] et al. in 1993 and Gibson and di Saint’Agnese[17] in 1963. This would occur because of the higher rate of sweat evaporation in the dry sauna than in the steam sauna where the relative humidity was approximately 100 %.[15] This means that the subject, in the dry sauna, would not need to sweat as much in order to cool down the body since the sweat evaporates more quickly than the sweat in the steam sauna. On the other hand, in the steam sauna, where relative humidity is high, the body could not cool down as quickly since the sweat on the skin could not evaporate into the saturated air. From the data that we obtained, we found that there was no significant difference between the

16 results from the dry sauna and the steam sauna trials for sodium and potassium ions. There was no observable trend between the two external conditions. The data sets from the steam sauna and the dry sauna for the potassium and sodium ions overlapped when concentration was graphed versus time (Figures 7 and Figure 9). A statistical analysis comparing the two physiological conditions was impossible because the number of trials was too small. Nonetheless, observation of these graphs will show that there is no distinction between the trials performed in the dry sauna and the trials performed in the steam sauna since there is so much overlapping between the plots. However, for the calcium ion, both trials in the dry sauna yielded higher concentrations than the trials in the steam sauna (Figure 8). This result contradicts our prediction that the ion concentration in the dry sauna should be less than that of the steam sauna. From Figure 8, we may believe that the difference in relative humidity affects the concentration of calcium in sweat, but further experimentation must be done to prove this correlation. Additionally, no previous literature indicates any significant concentrations of calcium in normal sweat. This prevents us from performing a historical comparison in order to verify the validity of our data. One important observation to be made from the potassium graph (Figure 9) is the fact that the first concentration point for potassium in each trial tends to be much higher than the rest. This type of characteristic was also noted by Sato and Sato in 1990.[18] The first data point represents the measurement of sweat during the first 15 minutes. During this time, the body is attempting to restore ion equilibrium and adjust to the environmental conditions. At temperatures above 37 C, the body needs to sweat in order to maintain its homeostatic internal conditions. Furthermore, the first sweat produced by the body contains many ions and impurities that tend to inflate the concentrations of the first set of data. This anomaly can also be explained physiologically. The ion pumps located in the basal and ductal sweat cells may not be completely active at the start of sweat excretion. The sodium-potassium pumps may not become active until the body begins to regulate its temperature. At this point, the ductal cells actively pump potassium from the extracellular fluid into the cell. This pump decreases the potassium concentration in the sweat for some time as correlated to the rapid decrease in potassium ion concentration between the 15 minute measurement and the 30 minute measurement as

17 shown in Figure 9. Concurrently, the sodium concentrations remain fairly constant in the sweat. Without any active pumping, the sodium concentrations should decrease over time because the body needs to retain sodium ions. The sodium ion concentrations remain fairly constant for two reasons. First, the sodium pumped into the sweat in exchange for potassium ions. Secondly, as sweat rate increases with time as shown by Boisvert,[15] the sodium ion concentration increases slightly.[15] It can not be firmly concluded that our sodium concentration increases over time, but it is evident that the sodium concentrations are constant over time whereas the other two ions show a drastic decrease in ion concentration between the 15 and 30 minute measurements. Regardless, the consistency of the data after the first 30 minutes for all ions justifies the conclusion that the first set of data is not completely representative of the data throughout the experiment. A major limitation of our experiment was the fact that we were only able to perform two trials for each physiological condition (dry and steam sauna) because of time constraints. Therefore, statistical analysis was very difficult and observing trends within trials are virtually impossible. Two different approaches were taken to compare the trials. First, we observed that the data points at 15 minutes were much higher than the other four points in the trial for potassium and calcium ions. For all three ions, there is a region on the graphs (Figures 7-9) where the concentrations are fairly constant. In order to treat the data as though the concentrations were not dependent on time (since the concentrations remained fairly consistent after 15 minutes), we excluded the data points at the first 15 minutes and used the other four points to find the means and perform a t- test analysis. This was also justified by the fact that sweat rate tends to become more constant after the body has had time to adjust to the external stimuli. Hence, we performed t-tests comparing the mean of one trial to the mean of the other trial within the same physiological condition. This data is listed in Table 2. From the Table, one can see that the data for the sodium ion and the potassium ion are statistically indifferent for both external conditions (dry sauna and steam sauna). Additionally, the data for concentration of calcium ion in the steam sauna seems to be statistically indifferent. However, for the calcium ion in the dry sauna, there is a statistical difference between trials. The t-stat value for these trials is 4.258 which is greater than the t-critical value of 2.447 for 95 %

18 confidence and n = 4. Secondly, we performed a unique statistical analysis comparing the two trials of data within each environmental condition. A method of comparing two trials regardless of their dependency on time was defined to analyze any consistency in the data. This method differed with the t-test method because it assumes that there was no time dependency on concentration and that the concentrations remained fairly constant. This method assumes that there may exist a strong relationship between time and concentration and tests this consistency between the trials. Specifically, the data for the concentration of a particular ion in trial one was plotted against the concentration of a particular ion in trial two. Ideally, if the two trials were identical to each other, it would be logical to conclude that these two trials were not statistically different. Therefore, time was ignored completely since, in an ideal situation, the time dependency for ion concentration would be the same for the two trials when run at identical conditions. If two sets of data within the same external condition (i.e. the steam sauna) yielded identical ion concentrations at every timed measurement, the graph of the ion concentration in trial two versus the concentration of the same ion in trial one would be perfectly linear with a R2 value of one. Once the graph of ion concentration for trial one versus trial two was made, a linear regression was performed, setting the y-intercept equal to zero. Figure 10 shows what an ideal graph of two trials of data would look like. We performed this statistical analysis, comparing trial one and trial two for each of the ions in each environmental condition. We evaluated the precision by observing the R2 value of the best-fit lines. Only the two trials for the calcium ion in the steam sauna showed consistency. Figure 11 shows that the R2 value of the linear regression between the trials’ data is 0.961. The low R2 values of the linear regressions for the rest of the data indicate that there were many other variables that caused variation between the two trials. Food and drink consumption in between the trials in addition to different pre-sauna dietary habits may have affected the consistency between trials. In order to improve the consistency in the future, it is necessary to perform sufficient trials under the same conditions in order to define a relationship between ion concentration and time. Another important aspect of this experiment was comparing the ion concentrations found in the sweat samples with those found in normal blood. As shown in Table 3, the normal ion concentration in blood of sodium, potassium, and calcium are

19 3243 ppm, 166.2 ppm, and 50.71 ppm, respectively.[6] Our experiment yielded mean concentration values for sodium and calcium for both conditions to be 1884 ppm and 8.441 ppm, respectively, which are well below the blood ion concentration levels mentioned above. This would be expected since the sweat cells would attempt to maintain ion equilibrium within the cells while excreting more dilute solutions of sweat to cool down the body. However, we found the ion concentration for potassium to be 236.0 ppm. This value was above the normal blood concentration for potassium. When one eliminates the first potassium data point, which was discussed to be an anomaly within the data, the mean potassium concentrations in both steam and dry sauna trials become comparable to that of blood concentration for potassium. However, the potassium ion concentration is still a bit high since it would normally be expected to have all ion concentrations less than that of blood. An explanation of this was briefly mentioned in the Background section. Since each ductal sweat cell has a sodium- potassium pump to regulate ion concentration in the body, an increase in the potassium ion will correlate with a decrease in the sodium ion concentration.[7] This would explain that sodium ion concentration was 44 % less than that of blood. As shown in Table 3, our potassium concentration was almost equal to that of blood while the sodium concentrations were well below the level in normal blood. Sato, Feibleman, and Dobson also previously observed this trend in 1970.[7] When one compares the ionic concentration of the sports drink, Gatorade Thirst Quencher, with the experimental values in this experiment, one finds that the sodium ion concentration of the drink is approximately a quarter of that found in the sweat while the potassium ion concentration of the drink is approximately half of that found in the sweat. This is an interesting observation because this sports drink’s purpose is to replenish any losses in fluids and ions. The reason why the sodium ion concentration may be so low is because smaller amounts of sodium are ideal for rapid fluid absorption. For the ionic concentration of potassium, there is not a clear explanation why it is so low in the drink. Perhaps there are industrial or economic reasons why Stokely-Van Camp, Inc. cannot increase the [K+] concentration or maybe they ran sweat studies solely on athletes, which may have sweat that is more diluted in ionic concentration than the general population.

20 The concentrations of the ions investigated in our experiment were also compared to those of literature values for sweat under other conditions. Table 4 shows the ion concentrations of sodium and potassium in sweat when isolated both pharmacologically and thermally. Drug induced sweat experiments are very common because of their efficiency and ability to control the degree of sweating by varying the concentrations of the drug. The most common drug used was pilocarpine.[7] Although pharmacologically induced sweat may have been easier to manipulate, the actual mechanism of sweat production tends to skew data. Since the drugs pacify the sodium-potassium pumps in the sweat cells, there was a higher overall sodium ion concentration in the sweat that was produced pharmacologically than that in sweat that was produced thermally. For our experiment, we only investigated the thermal effects of sweat production. The majority of research done on sweat ion concentration in the past has used the pharmacological approach in the collection of samples. Additionally, the drug-induced samples were collected from the forearm, which may have caused some deviation from their values and the ones that were collected in this experiment, but the main difference occurs because pharmacological effects tend to increase ion concentrations in sweat. However, one particular experiment performed by Taylor et al. found the median [K+] in sweat to be 648 ppm and the [Na+] to be 246 ppm when thermal effects were the only factors initiating sweat production.[16] These values are significantly lower than our experimental mean values for these ions. There are a few reasons for this. First, Taylor’s experiment entailed collection of sweat over a nine-hour period. Ion concentration by the end of the experiment would have been very low. Second, the ambient temperature was only slightly greater than body temperature at 32 C, which meant that the sweat rate was slow. Since the sweat rate was considerably slower than that of this experiment, it can be concluded that the difference in sweat rate was the main cause for the concentration discrepancy. From these varying values of concentration from many different sources, it can be concluded that ion concentrations in sweat changes considerably from one experiment to another. As a result, our data as well as data in the literature are not completely comparable. In order to improve sweat collection analysis for the future, many more trials must be performed under the same conditions, and all data from various experiments need to be pooled and directly compared.

21 ACKNOWLEDGEMENTS

Dr. Henry R. Drott, Chief Investigator for Cystic Fibrosis Sweat Collection at the Children’s Hospital of Philadelphia

Gia Hchevia, Biotechnologist and Researcher at Hospital of the University of Pennsylvania

22 REFERENCES

[1] Quinton, Paul M., “Physiology of sweat secretion”, Kidney International, Vol. 32, Suppl. 21 (1987), p. S-102 – S-108.

[2] Fox, Stuart Ira, Human Physiology, 5th ed., Wm. C. Brown Publishers: Boston. 1996. p. 16.

[3] http://www.thriveonline.com/health/Library/CAD/abstract20258.html

[4] Fox, Stuart Ira, Human Physiology, 5th ed., Wm. C. Brown Publishers: Boston. 1996. p. 12.

[5] Bijman, Jan, “Transport process in the eccrine sweat gland”, Kidney International, Vol. 32, Suppl. 21 (1987), p. S-109 – S-112.

[6] http://infonet.med.cornell.edu/lab/text.htm

[7] Sato, Kenzo, Cary Feibleman, and Richard L. Dobson, “The Electrolyte Composition of Pharmacologically and Thermally Stimulated Sweat: A comparative study”, The Journal of Investigative Deratology, Vol. 55, No. 6 (1970), p. 433-438.

[8] Fox, Stuart Ira, Human Physiology, 5th ed., Wm. C. Brown Publishers: Boston, 1996. p. 132.

[9] http://www.biology.washington.edu/bsa/ionTransport/atpdependentpumps

[10] Fox, Stuart Ira, Human Physiology, 5th ed., Wm. C. Brown Publishers: Boston, 1996. p, 136.

[11] http://www.gatorade.com/pages/gatorade/science.html

[12] http://www.gatorade.com/pages/gatorade/sci_label.html

[13] http://www.gatorade.com/pages/gatorade/sci_makesitwork.html

[14] Brisson, G. R., P. Boisvert, F. Péronnet, H. Perrault, D. Boisvert, and J. S. Lafond, “A simple and disposable sweat collector”, European Journal of Applied Physiology and Occupational Physiology, Vol. 63 (1991), p. 269-272.

[15] Boisvert, P., K. Nakamura, S. Shimai, G. R. Brisson, and M. Tanaka, “A modified, local sweat collector for warm and humid conditions”, European Journal of Applied Physiology and Occupational Physiology, Vol. 66 (1993), p. 547-551.

[16] Taylor, Richard P., Adrian A. Polliack, and Dan L. Bader, “The analysis of

23 metabolites in human sweat: analytical methods and potential application to investigation of pressure ischaemia of soft tissues”, Annals of Clinical Biochemistry, Vol. 31 (1994), p. 18-24.

[17] Gibson, Lewis E. and Paul A. di Saint’Agnese, “Studies of salt excretion in sweat”, The Journal of Pediatrics, Vol. 62, Number 6 (1963), p. 855-867.

[18] Sato, Kenzo and Fusako Sato, “Na+, K+, Cl-, and Ca2+ concentrations in cystic fibrosis eccrine sweat in vivo and in vitro”, Journal of Laboratory Clinical Medicine, Vol. 115, Number 4 (1990), p. (504-511).