madman
Super Moderator
Accurate Measurement and Harmonized Reference Ranges for Total and Free Testosterone Levels (2022)
Ravi Jasuja, Ph.D., Karol M. Pencina, Ph.D., Liming Peng, MSc, Shalender Bhasin, MB, BS*
INTRODUCTION
A central tenet of endocrinology is that the hormones secreted by the endocrine glands circulate and regulate the function of distant organs. The circulating concentration of the hormone often is used as a marker of the functional state of that endocrine gland; there is an optimum or healthy range of hormone concentration, and deviations from this range are associated with the disease. Thus, a diagnosis of clinical disorders associated with hormonal deficiency (eg, hypothyroidism) and hormonal excess (hyperthyroidism) is predicated crucially on accurate measurement of the circulating hormone concentration. Because hypogonadism in men is a syndrome characterized by symptoms and signs associated with consistently low testosterone levels, accurate and precise measurement of circulating testosterone concentration is an essential component of the diagnostic evaluation of patients suspected of testosterone deficiency.1 The ability to diagnose and treat testosterone deficiency correctly requires accurate and precise measurement of total testosterone levels and free testosterone levels, reliable reference ranges derived in relevant populations, and an understanding of the relationship between circulating testosterone concentrations and disease outcomes. The assay accuracy and rigorously derived reference ranges have an impact on the diagnosis and treatment of individual patients. The thresholds of hormone concentrations that distinguish healthy from diseased people in the general population influence the estimates of the disease prevalence and thereby health policy.
Circulating testosterone is bound mostly to human serum albumin (33% to 54%) and sex hormone-binding globulin (SHBG); the fraction of circulating testosterone bound to SHBG varies in men and women, with approximately 44% of testosterone bound to SHBG in men and 66% in women. A small fraction is bound to orosomucoid and cortisol-binding globulin; only 2% to 4% of circulating testosterone is unbound or free2 (Fig. 1). Conditions associated with elevated SHBG concentrations include old age, hyperthyroidism, polymorphisms in the SHBG gene, liver disease, human immunodeficiency virus infection, inflammatory conditions, and some medications, such as estrogens and some antiepileptics. SHBG concentrations are decreased with obesity, type 2 diabetes mellitus, polymorphisms in the SHBG gene, hypothyroidism, acromegaly, nephrotic syndrome, advanced liver disease, and treatment with androgens, progestins, and glucocorticoids.
Total testosterone refers to the sum of bound and unbound testosterone concentrations in the circulation. Only the unbound or free fraction can enter the cell and exert its biological effects. The term, bioavailable testosterone, refers to the fraction of circulating testosterone that is not bound to SHBG and connotes the view that testosterone binds to human serum albumin with low affinity and can dissociate readily in the tissue capillaries, especially in organs with long transit times, such as the liver and brain3,4 (see Fig. 1).
This article reviews the extant methods for the measurements of total testosterone concentrations and free testosterone concentrations, their relative merits and limitations, and the application of reference ranges to interpret the circulating concentrations of total testosterone and of free testosterone and offers an approach to increase diagnostic accuracy in the evaluation of men suspected of testosterone deficiency.
*METHODS FOR THE MEASUREMENT OF TOTAL TESTOSTERONE IN HUMAN SERUM OR PLASMA
Serum total testosterone concentrations can be measured using antibody-based immunoassays, such as radioimmunoassays, enzyme-linked immunosorbent assays, and immunofluorometric or immunochemiluminescent assays; aptamer-based assays; or mass spectrometry-based assays. The immunoassays utilize a high-affinity antibody to recognize and bind the analyte with a high degree of specificity in the human serum or plasma (Fig. 2). For detection of the antigen-antibody complexes, the immunoassays use a variety of labels that are linked to the antibody and sometimes to the antigen. Some of the earliest immunoassays used radioactive tracers as labels and were referred to as radioimmunoassays. Enzyme-linked immunosorbent assays (ELISAs) and enzyme-multiplied immunoassays use an enzyme, such as horseradish peroxidase, alkaline phosphatase, or glucose oxidase, linked to the primary or secondary antibody to produce a color change in the presence of the analyte that can be detected quantitatively. Chemiluminescence immunoassays use chemical probes that generate light emission during the analytical reaction.
The first-generation radioimmunoassays for testosterone had limited sensitivity and significant cross-reactivity of dihydrotestosterone and some other steroids. To overcome the dual problems of limited sensitivity and cross-reactivity of these early assays, a large volume of serum or plasma was extracted using organic solvents, such as methanol or hexane, and the steroidal extract of serum or plasma was subjected to chromatography on activated silica gel, high-pressure liquid chromatography, or another chromatographic procedure to separate testosterone from potentially cross-reacting steroids prior to radioimmunoassay. The assays that used extraction and chromatography had better sensitivity and avoided nonspecificity due to cross-reacting steroids and interference from plasma proteins. Because these extraction immunoassays were labor-intensive, platform-based assays were developed that eliminated the extraction and chromatographic steps to achieve high throughput and to reduce cost. Today, these platform-based direct immunoassays are widely used in clinical chemistry laboratories around the world because of their efficiency and relatively low cost but, as demonstrated by multiple studies,5,6 these direct immunoassays suffer from problems of nonspecificity, limited sensitivity, and high imprecision in the low range; therefore, they are not suitable for testosterone measurements in women, prepubertal children, and hypogonadal men.7,8 Wang and colleagues6 compared serum total testosterone levels measured using several commonly used automated and manual immunoassay methods with measurements performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and found that, in the low range, the immunoassays exhibited high levels of imprecision and inaccuracy and significant bias relative to an LC-MS/MS method.6
Aptamers are single-stranded nucleic acids that can bind the targeted analyte with high affinity and specificity. Aptamers can be synthesized in vitro, characterized for their target specificity and binding affinity using high throughput in selection methods, and immobilized to a matrix if needed.9–12 The aptamers offer several advantages over antibodies including their greater stability, longer shelf-life, their amenability to in vitro synthesis, modification, and immobilization.9–12 Few aptamer-based testosterone assays, however, are in clinical use at present.
LC-MS/MS has emerged as the method of choice with the highest specificity and sensitivity for the measurement of total testosterone in human serum or in plasma.8,13,14 LC-MS/MS assays typically involve initial extraction of the serum or plasma using either solid-phase extraction or liquid extraction using an organic solvent, followed by separation of compounds based on their polarity by high-pressure liquid chromatography.15,16 The eluted compounds are transferred to the mass spectrometer, where the analytes are ionized and separated based on their mass and charge. In tandem mass spectrometry, the precursor ions are transferred to a chamber where they are bombarded with a collision gas, such as nitrogen or xenon, and fragmented into product ions, and the mass of the product ions is quantified in the detector. Deuterated testosterone is added to each sample as an internal standard to correct for recovery. Gas chromatography-mass spectrometry provides even greater specificity than LC-MS/MS, but LC-MS/MS offers higher throughput. Consequently, LC-MS/MS assays have become widely available from many large commercial and some research laboratories; however, they require a greater initial investment in expensive equipment and a higher level of technical skill and remain more expensive than immunoassays. Unfortunately, despite their nonspecificity and imprecision in the low range, platform-based ELISAs and chemiluminescent immunoassays for testosterone continue to be used far more commonly than LC-MS/MS assays in hospital laboratories.
*METHODS FOR THE DETERMINATION OF FREE TESTOSTERONE CONCENTRATIONS
Free testosterone concentrations can be measured either directly using one of several available methods or estimated from total testosterone, SHBG, and albumin concentrations. The methods for the direct measurement of free testosterone include equilibrium dialysis,17,18 centrifugal ultrafiltration,19,20 steady-state gel filtration,21 flow dialysis,22 and direct tracer analog immunoassays.23 The equilibrium dialysis method employs dialysis of serum or plasma sample across a semipermeable membrane; the unbound testosterone crosses the dialysis membrane and equilibrates across the dialysis chambers whereas the protein-bound testosterone is retained on the sample side. The dialyzed fraction can be measured directly by LC-MS/MS; alternately, a tracer amount of radiolabeled testosterone can be added to the serum or plasma sample, and free testosterone concentration can be estimated by multiplying the percent of tracer in the dialyzed fraction with total testosterone concentration in the sample. The other methods, such as centrifugal centrifugation and gel filtration, use different techniques for separating unbound from bound testosterone. The equilibrium dialysis and centrifugal ultrafiltration methods have been shown to provide comparable results.18,19 Several laboratories offer the measurement of salivary testosterone24 as a marker of serum-free testosterone concentrations; however, salivary testosterone assays have not been shown to be an accurate measure of serum-free testosterone concentrations.24 Additionally, uneven sample desiccation, potential from contamination of oral contents and bacteria, and the paucity of rigorously derived reference ranges have prevented the wide adaption of salivary testosterone assays in clinical practice.
Equilibrium dialysis is considered the reference method against which all other methods are compared. Equilibrium dialysis methods are available from many large commercial laboratories but usually are not available in most local hospital laboratories. Furthermore, because of interlaboratory differences in assay procedures and lack of harmonized reference ranges, free testosterone should be measured in a reliable laboratory. Direct tracer analog methods are widely used in hospital laboratories; however, these assays are inaccurate and their use is not recommended.1,25,26
*Algorithms for Estimating Free Testosterone Concentrations
Because of the complexities in the current methods of measuring free testosterone, several equations have been published for the calculation of free testosterone concentrations from total testosterone, SHBG, and albumin concentrations.27–31 These equations can be broadly categorized into those that use the law of mass action equations27,29,31 and those that are deriving empirically using regression methods.30 The law of mass action equations, such as those published by Sodergard and colleagues,27 Vermeulen and colleagues,29 and Mazer,31 are based on the assumptions of linear binding of testosterone to SHBG with a fixed association constant. Furthermore, these equations assume that testosterone binds to a single binding site on human serum albumin with low affinity. Recent studies using modern biophysical techniques have shown that the binding of testosterone and estradiol to SHBG is a dynamic nonlinear process that involves an allosteric interaction between the 2 SHBG monomers, such that the Kd varies dynamically across the range of sex hormone and SHBG concentrations28,32 (Fig. 3). The estimates of free testosterone concentration using the novel Ensemble ALlstery Model match closely the concentrations measured derived by the equilibrium dialysis method.28 All algorithms are highly dependent on the accuracy and sensitivity of the total testosterone and SHBG assays. Furthermore, recent studies of testosterone’s binding to human serum albumin using 2-dimensional nuclear magnetic resonance and fluorescence spectroscopy have revealed the presence of multiple, allosterically coupled binding sites for testosterone on albumin33 (see Fig. 3). Testosterone shares these binding sites on human serum albumin with free fatty acids and many commonly used drugs, such as ibuprofen and warfarin, that could displace testosterone from human serum albumin under various physiologic states or disease conditions.33
*Bioavailable Testosterone
Bioavailable testosterone can be measured directly using the ammonium sulfate precipitation method that precipitates SHBG-bound testosterone or can be calculated from total testosterone, SHBG, and albumin. The high level of imprecision of the ammonium sulfate precipitation method and the lack of rigorously derived reference range, however, limit its utility in clinical practice.
*OPTIMIZING THE MEASUREMENTS OF TOTAL TESTOSTERONE AND FREE TESTOSTERONE TO REDUCE DIAGNOSTIC INACCURACY
The circulating concentrations of total testosterone and free testosterone vary substantially among men and in the same individual over time due to biological factors as well as measurement variation. A substantial fraction of the population-level variation in total testosterone levels among men is due to genetic factors, including genetic variants in the SHBG locus and on the X chromosome and some autosomes.34–36 Testosterone levels vary over time due to its pulsatile secretion and its circadian and circannual secretory rhythms. Testosterone is secreted in bursts approximately once every 90 minutes. Testosterone levels are higher in the morning than in the late afternoon; this diurnal rhythm is dampened in older men. The seasonal variation has been reported more clearly in geographic areas exposed to wide seasonal variation in temperature and daylight (eg, Northern Norway)37; in these regions, serum testosterone levels are the lowest in summer and the highest in early winter months. Testosterone levels decline after a meal, especially after a glucose load.38 Testosterone levels may be transiently suppressed during an acute illness; therefore, the evaluation of hypogonadism should be avoided during an acute illness.
Therefore, testosterone levels should be measured on 2 or more separate occasions within 4 hours to 5 hours of waking after an overnight fast.1 Total testosterone level should be measured using an accurate assay, preferably an LC-MS/MS assay, that is certified by an accuracy-based standardization or quality control program (eg, Centers for Disease Control and Prevention’s [CDC] Hormone Standardization Program for Testosterone [HoST]).1
Due to the complexities of free testosterone measurements and lack of rigorously derived reference ranges, an expert panel of the Endocrine Society recommended the use of total testosterone as the initial test for screening men suspected of testosterone deficiency.1 Free testosterone level should be measured in men with conditions associated with alterations in binding protein concentrations or in men in whom total testosterone concentrations are at or near the lower limit of the normal male range.1 Free testosterone concentration should be measured directly, preferably using an equilibrium dialysis assay in a reliable laboratory; if equilibrium dialysis assay is not available, free testosterone concentration should be estimated using an equation that provides a close approximation of values derived using the equilibrium dialysis method.1 The direct tracer analog assays for free testosterone assays should be avoided.8
*REFERENCE RANGES FOR TOTAL TESTOSTERONE LEVELS AND FREE TESTOSTERONE LEVELS
Reference Range for Total Testosterone in Men
The reference range refers to the distribution of the circulating concentration of a hormone or analyte in the general population and provides the foundational basis for distinguishing normal from low or high values.26Therefore, rigorously derived reference ranges are essential for establishing a diagnosis of hypogonadism. The authors and others have published reference ranges for circulating testosterone levels in the Framingham Heart Study (FHS) and in other populations.39–42 Because of differences in the study populations, assay methodology, and calibrators, however, reference ranges derived in 1 population may not be applicable to other populations or assays. Accordingly, under the auspices of the Endocrine Society, the authors compared the distribution of total testosterone concentrations in epidemiologic studies that included men from different geographic regions of the United States and Europe and generated consensus reference ranges for total testosterone levels in men.43Serum testosterone levels were measured in 9054 community-dwelling men who were participants in 1 of 4 cohort studies in the United States and Europe: FHS, European Male Ageing Study (EMAS), Osteoporotic Fractures in Men Study, and Male Sibling Study of Osteoporosis.43
The assays used to measure testosterone concentrations in the 4 cohorts were cross-calibrated by measuring testosterone levels in serum samples from approximately 100 men in each cohort in the CDC Clinical Reference Laboratory using an assay calibrated to higher-order reference materials and using serum-based reference materials as additional accuracy controls. Normalizing equations were generated using Passing-Bablok regression and used to generate harmonized values, from which standardized, age-specific reference ranges were derived. In healthy non-obese men, ages 19 years to 39 years; harmonized 2.5th, 5th, 95th, and 97.5th percentile values were 264 ng/dL, 303 ng/dL, 852 ng/dL, and 916 ng/dL (9.2 nmol/ L, 10.3 nmol/L, 29.5 nmol/L, and 31.8 nmol/L, respectively) (Table 1). The median value was 531 ng/dL (18.4 nmol/L).43 Age-specific harmonized testosterone concentrations in nonobese men as well as in the entire study population also were derived, by decades of age, and were similar across cohorts.43 These harmonized reference ranges can be applied to all assays and laboratories that are certified by the CDC HoST Program.1
The assay imprecision and substantial biologic variation in testosterone levels over time should be considered in interpreting the reference ranges, especially when the measured testosterone concentrations are at or within 2 SDs of the threshold that distinguishes normal and low values. A true value of 274 ng/dL (9.50 nmol/L) in an assay whose lower limit is reported as 275 ng/dL (9.53 nmol/L) has a 95% probability of being reported within 2 SDs on either side of the true value if the measurements were to be repeated. Therefore, the lower limit of the normal range should not be viewed as an absolute cutpoint. The risk of misclassification is high when the measured concentrations are at, just below, or just above the cutpoint.
*Reference Range for Free Testosterone
Harmonized reference ranges for free testosterone levels using equilibrium dialysis currently are not available, and clinicians have to rely on reference ranges provided by a laboratory. The populations included and the statistical methods used for generating these reference ranges are not published. Reference ranges for free testosterone concentrations estimated using the Ensemble ALlstery Model in male participants of the FHS have been published.28 The distribution of free testosterone concentrations was studied in a reference sample of healthy young male participants (ages 19– 40 years; N 5 434) of the FHS: Generation 3, who were nonobese and nonsmokers and who did not have a diagnosis of cancer, cardiovascular disease, diabetes mellitus, hypertension, or hypercholesterolemia.28,39 Only men with the wild-type SHBG (CC for rs6258 single nucleotide polymorphism) genotype, who constitute nearly 98% of the population, were included.34 The 2.5th percentile value for free testosterone in nonobese men, ages 19 years to 40 years (mean age 32.6 years), in the FHS Generation 3, was 114.6 pg/mL28; the corresponding 2.5th percentile values for free testosterone level in FHS broad sample (ages 19–90 years; mean age 49.3 years) and the EMAS (ages 40–79 years; mean age 60 years)44 were 69.7 pg/mL and 58 pg/mL, respectively. In the EMAS, the men with free testosterone levels more than 2 SDs below the mean of the reference sample (T score < 2) were at increased risk for having sexual symptoms and elevated LH associated. Several caveats should be considered when applying these reference ranges. The estimates of free testosterone concentrations are affected greatly by the method used to measure total testosterone and SHBG. The FHS population is predominantly white. The distribution of normative ranges generated in community-dwelling, healthy young men needs further validation in clinical populations and randomized trials. Efforts currently are underway to generate reference ranges for free testosterone concentrations using a standardized equilibrium dialysis method.
SYNOPSIS
Because hypogonadism is a syndrome characterized by a syndromic constellation of symptoms and signs in association with consistently low testosterone concentrations, accurate and precise measurement of total, and, if indicated, free testosterone concentration is necessary to establish its diagnosis. Variations in testosterone levels over time due to genetic factors, secretory rhythms, and effects of food, medications, age, obesity, and disease; imprecision and inaccuracy in the measurement of total testosterone concentrations and free testosterone concentrations; variations in binding protein concentrations; and suboptimal reference ranges contribute to diagnostic inaccuracies. The use of accurate assays, such as the LC-MS/MS for the measurement of total testosterone concentration in a laboratory certified by an accuracy-based program (eg, CDC HoST Program), multiple measurements of testosterone over time to confirm the diagnosis in fasting early morning samples, and measurement of free testosterone levels using the equilibrium dialysis method, when indicated, and the application of rigorously derived reference ranges can reduce the risk of disease misclassification and enhance diagnostic accuracy.
Ravi Jasuja, Ph.D., Karol M. Pencina, Ph.D., Liming Peng, MSc, Shalender Bhasin, MB, BS*
INTRODUCTION
A central tenet of endocrinology is that the hormones secreted by the endocrine glands circulate and regulate the function of distant organs. The circulating concentration of the hormone often is used as a marker of the functional state of that endocrine gland; there is an optimum or healthy range of hormone concentration, and deviations from this range are associated with the disease. Thus, a diagnosis of clinical disorders associated with hormonal deficiency (eg, hypothyroidism) and hormonal excess (hyperthyroidism) is predicated crucially on accurate measurement of the circulating hormone concentration. Because hypogonadism in men is a syndrome characterized by symptoms and signs associated with consistently low testosterone levels, accurate and precise measurement of circulating testosterone concentration is an essential component of the diagnostic evaluation of patients suspected of testosterone deficiency.1 The ability to diagnose and treat testosterone deficiency correctly requires accurate and precise measurement of total testosterone levels and free testosterone levels, reliable reference ranges derived in relevant populations, and an understanding of the relationship between circulating testosterone concentrations and disease outcomes. The assay accuracy and rigorously derived reference ranges have an impact on the diagnosis and treatment of individual patients. The thresholds of hormone concentrations that distinguish healthy from diseased people in the general population influence the estimates of the disease prevalence and thereby health policy.
Circulating testosterone is bound mostly to human serum albumin (33% to 54%) and sex hormone-binding globulin (SHBG); the fraction of circulating testosterone bound to SHBG varies in men and women, with approximately 44% of testosterone bound to SHBG in men and 66% in women. A small fraction is bound to orosomucoid and cortisol-binding globulin; only 2% to 4% of circulating testosterone is unbound or free2 (Fig. 1). Conditions associated with elevated SHBG concentrations include old age, hyperthyroidism, polymorphisms in the SHBG gene, liver disease, human immunodeficiency virus infection, inflammatory conditions, and some medications, such as estrogens and some antiepileptics. SHBG concentrations are decreased with obesity, type 2 diabetes mellitus, polymorphisms in the SHBG gene, hypothyroidism, acromegaly, nephrotic syndrome, advanced liver disease, and treatment with androgens, progestins, and glucocorticoids.
Total testosterone refers to the sum of bound and unbound testosterone concentrations in the circulation. Only the unbound or free fraction can enter the cell and exert its biological effects. The term, bioavailable testosterone, refers to the fraction of circulating testosterone that is not bound to SHBG and connotes the view that testosterone binds to human serum albumin with low affinity and can dissociate readily in the tissue capillaries, especially in organs with long transit times, such as the liver and brain3,4 (see Fig. 1).
This article reviews the extant methods for the measurements of total testosterone concentrations and free testosterone concentrations, their relative merits and limitations, and the application of reference ranges to interpret the circulating concentrations of total testosterone and of free testosterone and offers an approach to increase diagnostic accuracy in the evaluation of men suspected of testosterone deficiency.
*METHODS FOR THE MEASUREMENT OF TOTAL TESTOSTERONE IN HUMAN SERUM OR PLASMA
Serum total testosterone concentrations can be measured using antibody-based immunoassays, such as radioimmunoassays, enzyme-linked immunosorbent assays, and immunofluorometric or immunochemiluminescent assays; aptamer-based assays; or mass spectrometry-based assays. The immunoassays utilize a high-affinity antibody to recognize and bind the analyte with a high degree of specificity in the human serum or plasma (Fig. 2). For detection of the antigen-antibody complexes, the immunoassays use a variety of labels that are linked to the antibody and sometimes to the antigen. Some of the earliest immunoassays used radioactive tracers as labels and were referred to as radioimmunoassays. Enzyme-linked immunosorbent assays (ELISAs) and enzyme-multiplied immunoassays use an enzyme, such as horseradish peroxidase, alkaline phosphatase, or glucose oxidase, linked to the primary or secondary antibody to produce a color change in the presence of the analyte that can be detected quantitatively. Chemiluminescence immunoassays use chemical probes that generate light emission during the analytical reaction.
The first-generation radioimmunoassays for testosterone had limited sensitivity and significant cross-reactivity of dihydrotestosterone and some other steroids. To overcome the dual problems of limited sensitivity and cross-reactivity of these early assays, a large volume of serum or plasma was extracted using organic solvents, such as methanol or hexane, and the steroidal extract of serum or plasma was subjected to chromatography on activated silica gel, high-pressure liquid chromatography, or another chromatographic procedure to separate testosterone from potentially cross-reacting steroids prior to radioimmunoassay. The assays that used extraction and chromatography had better sensitivity and avoided nonspecificity due to cross-reacting steroids and interference from plasma proteins. Because these extraction immunoassays were labor-intensive, platform-based assays were developed that eliminated the extraction and chromatographic steps to achieve high throughput and to reduce cost. Today, these platform-based direct immunoassays are widely used in clinical chemistry laboratories around the world because of their efficiency and relatively low cost but, as demonstrated by multiple studies,5,6 these direct immunoassays suffer from problems of nonspecificity, limited sensitivity, and high imprecision in the low range; therefore, they are not suitable for testosterone measurements in women, prepubertal children, and hypogonadal men.7,8 Wang and colleagues6 compared serum total testosterone levels measured using several commonly used automated and manual immunoassay methods with measurements performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and found that, in the low range, the immunoassays exhibited high levels of imprecision and inaccuracy and significant bias relative to an LC-MS/MS method.6
Aptamers are single-stranded nucleic acids that can bind the targeted analyte with high affinity and specificity. Aptamers can be synthesized in vitro, characterized for their target specificity and binding affinity using high throughput in selection methods, and immobilized to a matrix if needed.9–12 The aptamers offer several advantages over antibodies including their greater stability, longer shelf-life, their amenability to in vitro synthesis, modification, and immobilization.9–12 Few aptamer-based testosterone assays, however, are in clinical use at present.
LC-MS/MS has emerged as the method of choice with the highest specificity and sensitivity for the measurement of total testosterone in human serum or in plasma.8,13,14 LC-MS/MS assays typically involve initial extraction of the serum or plasma using either solid-phase extraction or liquid extraction using an organic solvent, followed by separation of compounds based on their polarity by high-pressure liquid chromatography.15,16 The eluted compounds are transferred to the mass spectrometer, where the analytes are ionized and separated based on their mass and charge. In tandem mass spectrometry, the precursor ions are transferred to a chamber where they are bombarded with a collision gas, such as nitrogen or xenon, and fragmented into product ions, and the mass of the product ions is quantified in the detector. Deuterated testosterone is added to each sample as an internal standard to correct for recovery. Gas chromatography-mass spectrometry provides even greater specificity than LC-MS/MS, but LC-MS/MS offers higher throughput. Consequently, LC-MS/MS assays have become widely available from many large commercial and some research laboratories; however, they require a greater initial investment in expensive equipment and a higher level of technical skill and remain more expensive than immunoassays. Unfortunately, despite their nonspecificity and imprecision in the low range, platform-based ELISAs and chemiluminescent immunoassays for testosterone continue to be used far more commonly than LC-MS/MS assays in hospital laboratories.
*METHODS FOR THE DETERMINATION OF FREE TESTOSTERONE CONCENTRATIONS
Free testosterone concentrations can be measured either directly using one of several available methods or estimated from total testosterone, SHBG, and albumin concentrations. The methods for the direct measurement of free testosterone include equilibrium dialysis,17,18 centrifugal ultrafiltration,19,20 steady-state gel filtration,21 flow dialysis,22 and direct tracer analog immunoassays.23 The equilibrium dialysis method employs dialysis of serum or plasma sample across a semipermeable membrane; the unbound testosterone crosses the dialysis membrane and equilibrates across the dialysis chambers whereas the protein-bound testosterone is retained on the sample side. The dialyzed fraction can be measured directly by LC-MS/MS; alternately, a tracer amount of radiolabeled testosterone can be added to the serum or plasma sample, and free testosterone concentration can be estimated by multiplying the percent of tracer in the dialyzed fraction with total testosterone concentration in the sample. The other methods, such as centrifugal centrifugation and gel filtration, use different techniques for separating unbound from bound testosterone. The equilibrium dialysis and centrifugal ultrafiltration methods have been shown to provide comparable results.18,19 Several laboratories offer the measurement of salivary testosterone24 as a marker of serum-free testosterone concentrations; however, salivary testosterone assays have not been shown to be an accurate measure of serum-free testosterone concentrations.24 Additionally, uneven sample desiccation, potential from contamination of oral contents and bacteria, and the paucity of rigorously derived reference ranges have prevented the wide adaption of salivary testosterone assays in clinical practice.
Equilibrium dialysis is considered the reference method against which all other methods are compared. Equilibrium dialysis methods are available from many large commercial laboratories but usually are not available in most local hospital laboratories. Furthermore, because of interlaboratory differences in assay procedures and lack of harmonized reference ranges, free testosterone should be measured in a reliable laboratory. Direct tracer analog methods are widely used in hospital laboratories; however, these assays are inaccurate and their use is not recommended.1,25,26
*Algorithms for Estimating Free Testosterone Concentrations
Because of the complexities in the current methods of measuring free testosterone, several equations have been published for the calculation of free testosterone concentrations from total testosterone, SHBG, and albumin concentrations.27–31 These equations can be broadly categorized into those that use the law of mass action equations27,29,31 and those that are deriving empirically using regression methods.30 The law of mass action equations, such as those published by Sodergard and colleagues,27 Vermeulen and colleagues,29 and Mazer,31 are based on the assumptions of linear binding of testosterone to SHBG with a fixed association constant. Furthermore, these equations assume that testosterone binds to a single binding site on human serum albumin with low affinity. Recent studies using modern biophysical techniques have shown that the binding of testosterone and estradiol to SHBG is a dynamic nonlinear process that involves an allosteric interaction between the 2 SHBG monomers, such that the Kd varies dynamically across the range of sex hormone and SHBG concentrations28,32 (Fig. 3). The estimates of free testosterone concentration using the novel Ensemble ALlstery Model match closely the concentrations measured derived by the equilibrium dialysis method.28 All algorithms are highly dependent on the accuracy and sensitivity of the total testosterone and SHBG assays. Furthermore, recent studies of testosterone’s binding to human serum albumin using 2-dimensional nuclear magnetic resonance and fluorescence spectroscopy have revealed the presence of multiple, allosterically coupled binding sites for testosterone on albumin33 (see Fig. 3). Testosterone shares these binding sites on human serum albumin with free fatty acids and many commonly used drugs, such as ibuprofen and warfarin, that could displace testosterone from human serum albumin under various physiologic states or disease conditions.33
*Bioavailable Testosterone
Bioavailable testosterone can be measured directly using the ammonium sulfate precipitation method that precipitates SHBG-bound testosterone or can be calculated from total testosterone, SHBG, and albumin. The high level of imprecision of the ammonium sulfate precipitation method and the lack of rigorously derived reference range, however, limit its utility in clinical practice.
*OPTIMIZING THE MEASUREMENTS OF TOTAL TESTOSTERONE AND FREE TESTOSTERONE TO REDUCE DIAGNOSTIC INACCURACY
The circulating concentrations of total testosterone and free testosterone vary substantially among men and in the same individual over time due to biological factors as well as measurement variation. A substantial fraction of the population-level variation in total testosterone levels among men is due to genetic factors, including genetic variants in the SHBG locus and on the X chromosome and some autosomes.34–36 Testosterone levels vary over time due to its pulsatile secretion and its circadian and circannual secretory rhythms. Testosterone is secreted in bursts approximately once every 90 minutes. Testosterone levels are higher in the morning than in the late afternoon; this diurnal rhythm is dampened in older men. The seasonal variation has been reported more clearly in geographic areas exposed to wide seasonal variation in temperature and daylight (eg, Northern Norway)37; in these regions, serum testosterone levels are the lowest in summer and the highest in early winter months. Testosterone levels decline after a meal, especially after a glucose load.38 Testosterone levels may be transiently suppressed during an acute illness; therefore, the evaluation of hypogonadism should be avoided during an acute illness.
Therefore, testosterone levels should be measured on 2 or more separate occasions within 4 hours to 5 hours of waking after an overnight fast.1 Total testosterone level should be measured using an accurate assay, preferably an LC-MS/MS assay, that is certified by an accuracy-based standardization or quality control program (eg, Centers for Disease Control and Prevention’s [CDC] Hormone Standardization Program for Testosterone [HoST]).1
Due to the complexities of free testosterone measurements and lack of rigorously derived reference ranges, an expert panel of the Endocrine Society recommended the use of total testosterone as the initial test for screening men suspected of testosterone deficiency.1 Free testosterone level should be measured in men with conditions associated with alterations in binding protein concentrations or in men in whom total testosterone concentrations are at or near the lower limit of the normal male range.1 Free testosterone concentration should be measured directly, preferably using an equilibrium dialysis assay in a reliable laboratory; if equilibrium dialysis assay is not available, free testosterone concentration should be estimated using an equation that provides a close approximation of values derived using the equilibrium dialysis method.1 The direct tracer analog assays for free testosterone assays should be avoided.8
*REFERENCE RANGES FOR TOTAL TESTOSTERONE LEVELS AND FREE TESTOSTERONE LEVELS
Reference Range for Total Testosterone in Men
The reference range refers to the distribution of the circulating concentration of a hormone or analyte in the general population and provides the foundational basis for distinguishing normal from low or high values.26Therefore, rigorously derived reference ranges are essential for establishing a diagnosis of hypogonadism. The authors and others have published reference ranges for circulating testosterone levels in the Framingham Heart Study (FHS) and in other populations.39–42 Because of differences in the study populations, assay methodology, and calibrators, however, reference ranges derived in 1 population may not be applicable to other populations or assays. Accordingly, under the auspices of the Endocrine Society, the authors compared the distribution of total testosterone concentrations in epidemiologic studies that included men from different geographic regions of the United States and Europe and generated consensus reference ranges for total testosterone levels in men.43Serum testosterone levels were measured in 9054 community-dwelling men who were participants in 1 of 4 cohort studies in the United States and Europe: FHS, European Male Ageing Study (EMAS), Osteoporotic Fractures in Men Study, and Male Sibling Study of Osteoporosis.43
The assays used to measure testosterone concentrations in the 4 cohorts were cross-calibrated by measuring testosterone levels in serum samples from approximately 100 men in each cohort in the CDC Clinical Reference Laboratory using an assay calibrated to higher-order reference materials and using serum-based reference materials as additional accuracy controls. Normalizing equations were generated using Passing-Bablok regression and used to generate harmonized values, from which standardized, age-specific reference ranges were derived. In healthy non-obese men, ages 19 years to 39 years; harmonized 2.5th, 5th, 95th, and 97.5th percentile values were 264 ng/dL, 303 ng/dL, 852 ng/dL, and 916 ng/dL (9.2 nmol/ L, 10.3 nmol/L, 29.5 nmol/L, and 31.8 nmol/L, respectively) (Table 1). The median value was 531 ng/dL (18.4 nmol/L).43 Age-specific harmonized testosterone concentrations in nonobese men as well as in the entire study population also were derived, by decades of age, and were similar across cohorts.43 These harmonized reference ranges can be applied to all assays and laboratories that are certified by the CDC HoST Program.1
The assay imprecision and substantial biologic variation in testosterone levels over time should be considered in interpreting the reference ranges, especially when the measured testosterone concentrations are at or within 2 SDs of the threshold that distinguishes normal and low values. A true value of 274 ng/dL (9.50 nmol/L) in an assay whose lower limit is reported as 275 ng/dL (9.53 nmol/L) has a 95% probability of being reported within 2 SDs on either side of the true value if the measurements were to be repeated. Therefore, the lower limit of the normal range should not be viewed as an absolute cutpoint. The risk of misclassification is high when the measured concentrations are at, just below, or just above the cutpoint.
*Reference Range for Free Testosterone
Harmonized reference ranges for free testosterone levels using equilibrium dialysis currently are not available, and clinicians have to rely on reference ranges provided by a laboratory. The populations included and the statistical methods used for generating these reference ranges are not published. Reference ranges for free testosterone concentrations estimated using the Ensemble ALlstery Model in male participants of the FHS have been published.28 The distribution of free testosterone concentrations was studied in a reference sample of healthy young male participants (ages 19– 40 years; N 5 434) of the FHS: Generation 3, who were nonobese and nonsmokers and who did not have a diagnosis of cancer, cardiovascular disease, diabetes mellitus, hypertension, or hypercholesterolemia.28,39 Only men with the wild-type SHBG (CC for rs6258 single nucleotide polymorphism) genotype, who constitute nearly 98% of the population, were included.34 The 2.5th percentile value for free testosterone in nonobese men, ages 19 years to 40 years (mean age 32.6 years), in the FHS Generation 3, was 114.6 pg/mL28; the corresponding 2.5th percentile values for free testosterone level in FHS broad sample (ages 19–90 years; mean age 49.3 years) and the EMAS (ages 40–79 years; mean age 60 years)44 were 69.7 pg/mL and 58 pg/mL, respectively. In the EMAS, the men with free testosterone levels more than 2 SDs below the mean of the reference sample (T score < 2) were at increased risk for having sexual symptoms and elevated LH associated. Several caveats should be considered when applying these reference ranges. The estimates of free testosterone concentrations are affected greatly by the method used to measure total testosterone and SHBG. The FHS population is predominantly white. The distribution of normative ranges generated in community-dwelling, healthy young men needs further validation in clinical populations and randomized trials. Efforts currently are underway to generate reference ranges for free testosterone concentrations using a standardized equilibrium dialysis method.
SYNOPSIS
Because hypogonadism is a syndrome characterized by a syndromic constellation of symptoms and signs in association with consistently low testosterone concentrations, accurate and precise measurement of total, and, if indicated, free testosterone concentration is necessary to establish its diagnosis. Variations in testosterone levels over time due to genetic factors, secretory rhythms, and effects of food, medications, age, obesity, and disease; imprecision and inaccuracy in the measurement of total testosterone concentrations and free testosterone concentrations; variations in binding protein concentrations; and suboptimal reference ranges contribute to diagnostic inaccuracies. The use of accurate assays, such as the LC-MS/MS for the measurement of total testosterone concentration in a laboratory certified by an accuracy-based program (eg, CDC HoST Program), multiple measurements of testosterone over time to confirm the diagnosis in fasting early morning samples, and measurement of free testosterone levels using the equilibrium dialysis method, when indicated, and the application of rigorously derived reference ranges can reduce the risk of disease misclassification and enhance diagnostic accuracy.
