Materials and Methods - Saddleback College



The Mean Testosterone Level is Higher in Adult Males With Deeper VoiceBen Hoffman, Sid Mirgati, Tim TurnbaughDepartment of Biological ScienceSaddleback CollegeMission Viejo, CA 92692 Testosterone is an anabolic steroid hormone in males that has variety of responsibilities including fetal development of genitalia and physical maturation during puberty; thus it can be hypothesized that testosterone levels are directly related to the deepness of voice in human males. To test this, a Salimetrics kit was used to measure the amount of free testosterone in subjects’ saliva. A micro-plate reader was used to provide a quantified testosterone concentration reading. The voice and saliva of ten (n=10) adult males was collected. Testosterone levels fluctuate throughout the day, thus multiple samples were collected and the mean value of the samples was used in calculations. One set of the lowest note a subject could produce was also collected. After the analysis, the mean voice frequency of all subjects was measured and the samples were divided into two groups. The first group included the subjects with lower voice frequency than the total average frequency. The second group included the subjects with higher voice frequency than the total average frequency. The mean testosterone level of first group was 626.139 pg/mL (S.E.M ±134.728 pg/mL, C.L. ±428.766). The mean testosterone level of second group was 475.896 pg/mL (S.E.M ±35.546 pg/mL, C.L. ±91.375). To compare the mean testosterone value of each group, a one-tailed unpaired t-test was performed (P=0.179). The results rejected the hypothesis and stated there is no significant correlation between the testosterone level and voice frequency in adult males. IntroductionTestosterone - a key hormone present in every man - is responsible for many functions and developments in the human male. It is responsible for fetal development of the male genitalia, the physical changes that occur during male puberty, and a variety of other functions in the adult male, including sperm production, erections,?sex drive, muscle tone, and bone health (Walker, et al.,1997). In men, 95% of testosterone is created in the testes and the rest is produced in the adrenal glands. There are two major forms of testosterone in human body: bound testosterone and free testosterone. Bound testosterone is attached to another substance such as globulin or albumin. When the testosterone is attached to another molecule, it is modified and subsequently cannot fit into a receptor site. Since it cannot become active, it is eventually excreted by the body. Free testosterone is the amount of testosterone that floats through the blood on its own and is readily available for use by the body. In men, 35 to 155 pg/mL (picograms per milliliter) is the normal range of free testosterone. There may be daily variations as male testosterone levels?peak in the morning and then steadily decline until late evening. Stress, lack of sleep and many other factors can affect testosterone levels (Meuser, 1977).When getting the testosterone levels tested, the results may be misleading. If the level of bound testosterone is within normal range, it is not necessarily an indication of healthy levels. The level of free testosterone is more important, because free testosterone is the one that is more readily used by the body (Dabbs, Mallinger, 1999).Taking steroid hormones such as testosterone for performance enhancement in males can cause testicles to shrink and breast tissue to growth. For women, it can cause a deepened voice, an enlarged clitoris, hair loss from the head, and hair growth on the body and face. In both genders, steroid abuse can cause acne, mood swings, aggression, and other problems. A simple error in diet, such as eating a lot of sugar or high-glycemic foods, restricting fat, being abstaining from meat, or not getting enough magnesium, zinc, or vitamin D may lower testosterone levels. These lowered testosterone levels may affect the functions of individual development, immune system, libido, heart health, energy, weight control and emotional well-being (Apicella, Feinberg 2009). In men, the different depths of voice are influenced by different concentrations of circulating sex hormones and also by the androgen sensitivity of the target organs (Meuser, 1977). According to a Georgia State University experiment, salivary testosterone levels were “significantly associated with lower pitched voices among males but not among females (Dabbs, 1999).” Harries performed a similar study looking at how testosterone level affects the males voice during puberty. Their study concluded that testosterone was not “predictive of the changes, (but) there was a correlation with testis volume and testosterone levels." Alternative test methods, including multiple saliva samples from the Human Cognitive Neuroscience Unit of the School of Psychology and Sport Sciences, indicated even greater magnitude of the relationship between high testosterone and lower frequency. Some have hypothesized lower voices have had an evolutionary benefit of attracting females by demonstrating a high level of testosterone. This has been debunked, however, as a Harvard study demonstrated that while men prefer higher pitched women, women express no preference for the reverse on males (Apicella, 2009). Our study tests the correlation between the testosterone levels and the depth of voice in men. Since the testosterone fluctuates considerably from one time to the next we decided to collect multiple saliva samples and use the average of testosterone levels for the calculations. Based on past results, there is testosterone in saliva, and the average levels detected were 295 ± 36 pg/ml in adult males (Dabbs, Mallinger, 1999). Our researchers hypothesize that the mean testosterone level is going to be higher in men with lower voice frequency.Materials and MethodsTen adult subjects were used in this study. Subjects were adult male students at Saddleback College, Mission Viejo, California. All measurements were made on the 20th of November 2013. Subjects were contacted before and asked to participate in the study. Each participant signed an experiment specific agreement waiver and filled out a short questionnaire. After agreement was reached, we scheduled the day we would collect the saliva and voice samples from the subjects.On the scheduled day (November 20, 2013) the subjects were all present for the first set of sample-collecting between 8:20 to 8:40 am. Each subject was given a saliva collection aid and a pre-labeled collection vial. Subjects were asked to insert the ribbed-end of the saliva collection aid in the pre-labeled collection vial and allow saliva to pool in mouth before forcing the saliva through the saliva collection aid into the vial and fill approximately half the vial (1 mL). Then the saliva collection aid was removed and discarded and the cap was attached to the collection vial. It was made sure that the subjects passively drooled into their labeled collection container. Directly after saliva retrieval the researchers placed the test containers into a cold storage filled with ice to freeze the samples preventing degradation. All the voice sample-collecting occurred outside room SM 244 at Saddleback College, Mission Viejo, California. Subjects were asked to make the lowest note they were able to produce for 3 seconds. This was recorded using an iPhone 4S which yielded a collection rate of 44600 samples per second. The sample-collecting occurred when no other individual (except the subject himself and the researcher that was holding the recording device) was in approximately 5 yards of the subject to minimize the background noises in the recordings as much as possible. The second set of samples was collected under identical circumstances between 11:20 and 11:40 am. The second set of saliva samples was also placed into a cold storage filled with ice to freeze the samples preventing degradation. During the sample-collecting, each sample was labeled as a number in order to keep subject's privacy. In between collection and analysis the samples were stored in a freezer at -4 degrees Celsius. The analysis of testosterone samples occurred on November 22, 2013 in room SM 244 at Saddleback College, Mission Viejo, California. The researchers brought out all of the sample containers from the freezer at 8 am so the samples would be thawed in time for analysis. The reagents were also removed from the refrigerator the day before in order to come to room temperature. The Salimetrics preparation and analysis procedure was followed providing us with our raw data. Voice recordings were analyzed using Speech Analysis Software (SIL International, Dallas, Texas) to find the fundamental frequency (average pitch of the sample) of the subject's voice. The equation used by the Speech Analysis Software is a modified version of Cepstrum analysis Ffundamental=Cx=F-1{log?(|Fft|2}2 where represents a fourier transformation of the equation. The results of this equation changed the wave form into a more linear function making a calculation of average frequency possible within the Speech analysis software. ResultsThe data was run through a Microsoft Office excel spreadsheet. The program took the input data obtained from the microreader and outputted a 4- Parameter Sigmoid Minus Curve. The curve was adjusted to fit along the plot points of the standards as best as possible (minimum asymptote: 0.1, steepness -1.2, inflection point 100, and maximum asymptote 1). This curve allows the calculation of the testosterone concentration in the unknown saliva samples as demonstrated in Figure 1. Figure 1. Graph relating the Percent Bound of Standards and Unknowns with the Log of Testosterone Concentration (n=10). As the log of concentration increases, the percent bound for standards and unknowns decreases. The minimum asymptote was 0.1, the steepness was -1.2, the inflection point was 100, and the maximum asymptote was 1.After the testosterone concentrations for each well was gathered, they were arranged according to test and subject. The mean value of testosterone levels for each subject was calculated using: i=1Nti2N=Mean TestosteroneThe mean value of the voice frequency level from all subjects (n=10) was calculated 87.2 Hz.The subjects were separated into two groups: the first group includes the subjects with lower voice frequency than the total mean. The second group includes the subjects with the higher voice frequency than the total mean. For each group, the mean of testosterone level and voice frequency was obtained and analyzed separately.For the first group (subject with lower voice frequency than the total average) the mean testosterone level was 626.139 pg/mL (S.E.M ±134.728 pg/mL, C.L. ±428.766). For the second group (subject with higher voice frequency than the total average) the mean testosterone level was 475.896 pg/mL (S.E.M ±35.546 pg/mL, C.L. ±91.375). These data are shown in Figure Two. Figure 2. Mean Testosterone Concentration level compare to the Mean Frequency of Voice in each subject (n=10). The average voice frequency was measure 87.2 Hz. There was no significant difference in the mean testosterone level of subjects with voice frequency of below total average in compare to the subjects with higher voice frequency of the total average. (p=0.179, one-tailed unpaired t-test). Error Bars are mean ± SEM.Based on the p value (P=0.179), we can reject our hypothesis and accept the null hypothesis that there is no significant difference in the mean testosterone level in relation to fundamental voice frequency. Two other studies also concluded testosterone levels have no significant relationship to voice frequency in men (Hughes et .l, 1997) (Meuser and Nieschlaq, 1977). In our experiment the testosterone levels in subjects were significantly higher than the normal adult males. The possible explanation for this difference is the performance errors during the saliva preparation that could cause the incorrect ratio of the sample to reagent. This error could lower the number of reactions between the antibodies and the testosterone in Salimetric plates and eventually demonstrate higher testosterone level than the actual level.CitationsApicella, Coren and David Feinberg. 2009. Voice Pitch Alters Mate-Choice-Relevant Perception in Hunter-Gatherers. Proceeding of the Royal Society. Vol. 276:1077-1082.Dabbs, James and Alison Mallinger. 1999. High Testosterone Levels Predict Low Voice Pitch Among Men. Science Direct. Vol. 27, Issue 4. Evans, Sarah, Nick Neave, Delia Wakelin, and Colin Hamilton. 2008. The Relationship Between Testosterone and Vocal Frequencies in Human Males. Science Direct. Vol. 93, Issue 4-5.Hughes, J M Walker, D M Williams, and S Hawkins. 1997. Changes in the Male Voice at Puberty. Archives of Disease in Children. 77:445-447.Meuser W. Nieschlaq. 1977. Sex hormones and depth of voice in the male (Article in Germany). Deutsch Med Wochenschr. 25:102(8):261-4. ................
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