FINAL VERSION OF LAB REPORT (1)
- 1. Effect of Different Solutions on Urine Specific Gravity, Urine Volume, and pH Emerson Eggers 027:132:A01 4/29/14 Abstract: In this experiment, the subjects were randomly selected to different solution conditions. The solutions included: water, isotonic NaCl (300mOsm/L), hypertonic NaCl (500mOsm/L), cranberry juice, and a bicarbonate solution (500mOsm/L). Urine samples were obtained both prior to consumption of the solutions, and 60-75 minutes post-consumption. Urine specific gravity and pH were recorded at both time points, and urine volume was measured post-consumption. The results indicate that subjects in the water condition experienced a significant decrease in specific gravity, while subjects who received either of the NaCl solutions experienced a significant increase in specific gravity. When comparing the specific gravity of the two NaCl solutions, there was no significant difference. Subjects in the water condition also experienced a significantly higher urine volume, while the subjects who received either NaCl solution tended to have a lower urine volume. There was no significant difference in urine volume between the isotonic and hypertonic NaCl solutions. We attribute these results to the insertion and or removal of aquaporins in the collecting ducts by the antidiuretic hormone (ADH). The subjects who received the bicarbonate solution experienced a significant increase in urine pH, while subjects who received the isotonic NaCl solution or
- 2. cranberry juice experienced a decrease in pH. These results suggest that the kidneys adapts to changes in acid-base balances in the body by manipulating the amount of H+ or HCO3- ions which are: filtered, secreted, reabsorbed, and excreted. Introduction The renal system is one of the most intricate systems in the human body, and being so, it is also one of the hardest systems to comprehend. The renal system is involved in more than simply urination, and it plays a role in numerous processes in the body, all working together to maintain homeostasis. The kidneys work to maintain fluid balance, osmotic pressure, electrolyte balance, and pH. The kidneys also work to prevent the buildup of metabolic waste products in the body, which if not excreted, can reach toxic levels, and heighten the risk for renal faliure. This lab was performed in order to provide people with an insight into how urine samples can be used to assess renal function, and to discover how different solutions can affect variables such as urine specific gravity, urine volume, and urine pH. The main structures of the renal system include the kidneys, which are two bean shaped structures located in the back of the abdomen. The kidneys contain approximately one million nephrons, which are the functional units of the kidneys. The nephrons are ultimately in charge of regulating all of the homeostatic variables such as fluid volume, osmolarity, and pH by way of excretion. Excretion refers to the removal of a substance from the body via urine, and is dependent upon filtration, secretion, and reabsorption. Each of these processes is highly intricate, and are
- 3. greatly influenced by renal blood flow as well as hormones such as the antidiuretic hormone (ADH), Aldosterone, Renin, and Angiotensin. In this experiment, 60 subjects provided urine samples at two time points. The first urine samples were taken prior to the consumption of a randomized solution, and were tested for urine specific gravity and urine pH. The second urine samples were collected 60-75 minutes after the consumption of a randomized solution, and were once again tested for urine specific gravity and urine pH, as well as urine volume. Our expectation was that subjects in the water condition would experience a decrease in urine specific gravity as well as a high urine volume. We also expected the subjects who received either the isotonic or hypertonic NaCl solutions to experience an increase in specific gravity and a low urine volume. Finally, we expected the subjects who received the bicarbonate solution to experience an increase in urine pH, while the effects of isotonic NaCl (300mOsm/L) and cranberry juice were lesser known. Methods Experiment 11.1: Effect of Fluid Type on Urine Specific Gravity and pH This experiment involved a total of 60 college-aged students (37 male; 23 female). The experiment took place at approximately 2:00pm at the Field House on the University of Iowa campus. In order to complete the urinalysis, we used two common methods to assess both urine specific gravity and urine pH. An optical refractometer was used in order to examine urine specific gravity. This device was calibrated prior to testing, and works by measuring the refractive index of a urine sample compared to distilled
- 4. water (SG=1.000). Urine pH was examined using a pH meter, which measures the acidity or alkalinity of the urine samples. Each of these tests were performed by students not participating in the experiment, who had been informed on the protocol of each test prior to the experiment. Prior to their arrival at the testing facility, the subjects were instructed to follow a strict food/fluid protocol. This protocol included: being properly hydrated prior to the experiment, refraining from eating or drinking for 4 hours prior to the experiment (water was okay up to 2 hours prior to the experiment), and voiding their bladders 1 hour prior to the experiment. Upon their arrival to the classroom, the subjects randomly drew cards in order to be assigned to one of the following solution conditions: Plain water (500mL) Isotonic NaCl, 300mOsm/L (500mL) Hypertonic NaCl, 500mOsm/L (500mL) Cranberry Juice (500mL) Bicarbonate solution, 500mOsm/L (500mL) This experiment involved the collection of two urine samples from the subjects. The first urine sample was collected prior to the consumption of their specified solution. These urine samples were analyzed for both urine specific gravity and urine pH. Urine specific gravity was measured by saturating the glass of the optical refractometer with a urine sample, and then recording the specific gravity of that sample as shown on the refractometer (typical values range from 1.003 gm/cm^3 to 1.035 gm/cm^3). Once a samples specific gravity was measured, the
- 5. urine samples pH was measured through the use a pH meter. The results for each subject were then recorded in a pre-consumption column on an excel spreadsheet by another student not participating in the experiment. After the data from the first urine samples was recorded, each of the subjects consumed their specified solution. After 60-75 minutes, the subjects were instructed to provide another urine sample. This urine sample was provided by way of a glass beaker however, as urine volume was also measured in the post-consumption trial. After urine volume was recorded, an adequate sample was taken from each of the beakers in order to assess urine specific gravity and pH. Urine specific gravity and urine pH followed the same protocol as the pre-consumption tests. The results on urine volume, urine specific gravity, and urine pH were once again recorded on an excel spreadsheet. In order to limit confounding variables and assure accurate results, the following steps were also taken: Subjects used restrooms within a close proximity to the classroom to provide their urine samples Subjects were instructed not to collect the initial stream of urine for testing, as the initial stream may have contained contaminants The optical refractometer was cleaned with distilled water and a non- abrasive cloth after each test The pH probe was rinsed with distilled water and recalibrated in a buffer solution after each test
- 6. Statistical analysis was possible for this experiment due to the number of subjects participating in the experiment. We sought to examine how our dependent variables (urine specific gravity, urine pH, urine volume) changed with our independent variable (fluid consumed). Both a repeated-measures design and a between groups design were used in this experiment. Urine specific gravity and urine volume were assessed between water, isotonic NaCl (300mOsm/L), and hypertonic NaCl (500mOsm/L) by running a between groups ANOVA test, which resulted in a p-value under 0.05, so a post-hoc with an unpaired t-test was then run. In order to assess how specific gravity changed over time, a repeated measures paired t-test was also run for this comparison. Urine pH was assessed between isotonic NaCl (300mOsm/L), cranberry juice, and a bicarbonate solution (500mOsm/L). In order to assess how our dependent variable (pH) changed with the dependent variable (fluid type), a between groups ANOVA and post-hoc with an unpaired t-test was run. A repeated measures paired t-test was also performed in order to assess how pH changed between the two time points. Results Experiment 11.1: Effect of Fluid Type on Urine Specific Gravity and pH Specific gravity was shown to be significantly different between conditions (F2,36=16.15, p<0.05). A post-hoc with an unpaired t-test revealed that there was a difference between water and isotonic NaCl, as well as water and hypertonic NaCl (p<0.0167). There was no significant difference between isotonic NaCl and hypertonic NaCl (p=0.32, dof=2, t11=2.72 and 2.82 respectively). A paired t-test
- 7. showed a significant difference between the three conditions across the time points. Water (t11=3.65, p<0.05), isotonic NaCl (t11=2.72, p<0.05), hypertonic NaCl (t11=2.82, p<0.05). Figure 1 There was a significant difference in urine volume between the conditions (F2,33=79.05, p<0.05). Post-hoc testing with an unpaired t-test revealed that subjects in the water condition had the highest mean urine volume (p<0.0167). There were no other significant differences. Figure 2 0.980 0.990 1.000 1.010 1.020 1.030 Water 300 mOsm NaCl 500 mOsm NaCl SpecificGravity Mean Specific Gravity Preand PostConsumption of Three DifferentBeverages Pre-Consumption Post-Consumption Columns represent mean specific gravity +/- standard deviation (n=12) 0 50 100 150 200 250 300 350 400 Water 300 mOsm NaCl 500 mOsm NaCl Volume(mL/hr) Mean Urine VolumePost-Consumption of Three DifferentBeverages Columns represent mean urine volumes +/- standard deviation (n=12)
- 8. A significant difference was found to exist between the conditions for urine pH (F2,33=74.30, p<0.05). A post-hoc with an unpaired t-test revealed that the bicarbonate solution resulted in a higher pH than the isotonic NaCl (300mOsm/L) and cranberry juice solutions (figure 3). A paired t-test revealed that a difference existed between the two time points for each of the conditions. Isotonic NaCl (t11=3.76, p<0.05), bicarbonate (t11=9.47, p<0.05), cranberry juice (t11=4.24, p<0.05). No other differences were found to exist. Figure 3 Discussion Experiment 11.1: Effect of Fluid Type on Urine Specific Gravity and pH In order to assess the effects of different solutions on urine specific gravity and urine volume, we compared the results from the following conditions: water, isotonic NaCl (300mOsm/L), and hypertonic NaCl (500mOsm/L). The subjects in the 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 300 mOsm NaCl 500 mOsm NaHCO3 Cranberry UrinepH Mean Urine pH Preand Post Consumption of Three DifferentBeverages Pre-Consumption Post-Consumption Columns represent mean urine pH +/- standard deviation (n=12)
- 9. water condition experienced a decrease in specific gravity between the two time points, as well as a significantly higher mean urine volume in the post-consumption test compared to subjects in either of the NaCl conditions. We attribute this result to the fact that water normally stays within the ECF compartment upon consumption, thus diluting the plasma, and lowering its osmolarity. This decrease in plasma osmolarity is detected by hypothalamic osmoreceptors that are very sensitive to changes in osmotic pressures. These osmoreceptors proceed to decrease their rate of firing, which ultimately leads to the inhibition of the antidiuretic hormone (ADH), the primary hormone involved in the reabsorption of water. In the absence of this hormone, the medullary collecting duct is impermeable to water, and little water is reabsorbed. As seen in figure 1, the final result is a large volume of highly diluted water. The subjects in the isotonic and hypertonic NaCl conditions experienced an increase in specific gravity after the consumption of their solution (figure 1). This can be attributed to the fact that these solutions contained solutes within them (Na+, Cl-) that would work to increase the plasma osmolarity in the body. This increased plasma osmolarity triggers an increase in the firing of the osmoreceptors in the hypothalamus, and subsequently the secretion of ADH from the posterior pituitary gland. ADH exerts its effect on the endothelium of the collecting ducts, thereby inserting water channels (aquaporins) into these tissues, which ultimately allows for the reabsorption of water until the osmotic equilibrium is reached (1400mOsm/L). As evidenced in our results, the final outcome for this reaction is a small, very concentrated urine.
- 10. In clinical terms, pH (potential of hydrogen) is a measure of the acidity or alkalinity of a solution, in this case urine. It is measured on a scale from 0-14 (7=neutral, under 7=acidic, above 7=basic). Ideally, the optimum range for blood pH is between 7.35-7.45, and anything outside of this range can have dire consequences including the possibility of death if blood pH falls below 6.8 for even a matter of seconds. It is important to remember that blood pH is different than urine pH, and while urine pH can fluctuate enormously throughout a day, these fluctuations in urine pH are one of the mechanisms in which assures blood pH stays within its homeostatic range. Through the examination of these urine samples, we were able to see how each subject’s renal system was working to maintain acid-base balances. Experiment 11.1 also examined the effect of isotonic NaCl (300mOsm/L), cranberry juice, and a bicarbonate solution (500mOsm/L) on urine pH. The isotonic NaCl (300mOsm/L) was the control group in this experiment. The subjects who consumed the bicarbonate solution experienced a significant increase in urine pH when compared to the subjects in the isotonic NaCl or cranberry juice conditions. Bicarbonate is the most important renal mechanism for regulating acid-base balance, in addition to being the most important buffer in controlling the free H+ ion concentration in the ECF. The premise behind why the subjects who consumed the bicarbonate solution experienced an increase in pH is due to the fact that an excess of HCO3- molecules were present in the ECF after the consumption of the solution. The amount of HCO3- in the ECF now outnumbered the amount of free H+ ions, so that although some of the HCO3- did combine with the free H+ ions to form carbonic acid, not all of the HCO3- was used up in this reaction, and the excess HCO3- was
- 11. excreted in the urine. This HCO3- in the urine resulted in the increase in urine pH seen in table 3. With an increased acid load, such as in the cranberry condition, there is an increase in H+ ion concentration in the ECF. Since H+ ions are not easily able to be excreted, they depend upon buffers in the urine. Normally, phosphorous would be the primary buffer in this reaction, however phosphorous is highly dependent upon dietary intake, and not acid-base balance, so the supply of available phosphorous is used up quickly in this reaction. Vital to the excretion of H+ ions is the phenomenon of increased ammonium production in response to an increased acid load. The ammonium is produced in the proximal tubule, where it is then reabsorbed into the thick ascending limb of the medullary interstitium, and finally pumped into the collecting tubule, where it becomes ammonia (NH4+). While that process is occurring, H+ ions are being secreted into the tubular fluid of the collecting ducts, where they combine with the ammonia, to finally be excreted in the urine. In all, this process works to decrease urine pH by way of H+ ion excretion as well as HCO3- reabsorption via ammoniagensis. Due to the fact that the isotonic NaCl (300mOsm/L) was our control group in this experiment, we will not go into the details about its mechanism of action. However, it should be noted that the subjects who consumed the isotonic NaCl (300mOsm/L) experienced a decrease in urine pH, which can be attributed to dilutional acidosis. This experiment could be improved by performing the urine tests upon waking up in the morning. In the morning, urine is more concentrated due to the
- 12. lack of water consumption throughout the night, so any abnormal result may become more obvious. It may have also been interesting to perform a microscopic examination on the urine samples. This would have allowed us to observe if there were any irregularities in the urine samples, such as white blood cells that would indicate a possible infection.