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.
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 ppmCalcium Reference Range / 48.50 ppm – 52.91 ppm
Potassium Reference Range / 136.85 ppm – 195.5 ppm
Table 1: Ion Content of Blood[6]
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] uses the energy derived from ATP to drive the 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 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 GatoradeÒ stimulates people to drink more fluid voluntarily until the body is rehydrated.[13]
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 piece of permeable laboratory film paper, such as 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 mm thick polypropylene and sealed around the edges with surgical tape. After collection, the cellulose pad was placed in a tube and 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 3000Ô 20-200 mL and 100-1000 mL pipettes
· Fisherbrand® Redi-TipÔ 200-mL and 1000-mL 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
· FisherbrandÔ 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
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 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 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 each ion that will be tested before each run with 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 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 the each sweat sample so if we ran the sample through the AA spectrophotometer without diluting it, we would have lost all of our sample in that one measurement. Secondly, the sweat sample was too concentrated to fall into the linear range of our calibration curves.