Elsevier

Steroids

Volume 76, Issues 1–2, January 2011, Pages 48-55
Steroids

Accuracy of calculated free testosterone differs between equations and depends on gender and SHBG concentration

https://doi.org/10.1016/j.steroids.2010.08.008Get rights and content

Abstract

Serum free testosterone (fT) concentrations are often calculated, however different equations often yield discrepant results. This study explores the sources of this variability. We compared three established and two new equations that differed only by their testosterone association constants with isotope dilution equilibrium dialysis in two patient groups with different gender distributions. Equation components were examined to determine how they impacted correlation with isotope dilution equilibrium dialysis. Association constants derived for each patient group correlated best with isotope dilution equilibrium dialysis for that group and not the other set. Samples with the poorest correlation between isotope dilution equilibrium dialysis and calculated fT results had significantly higher SHBG concentrations. Regardless of equation, ≥25% of samples showed unacceptable deviation from isotope dilution equilibrium dialysis. Association constants and gender makeup and SHBG concentration of the patient groups used to establish an equation all significantly impact correlation with isotope dilution equilibrium dialysis. Application of many fT equations to wider populations will therefore frequently yield results that differ substantially from isotope dilution equilibrium dialysis.

Research highlights

▶ fT equations are affected by testosterone association constants, gender, and SHBG. ▶ Patient group-derived association constants may not perform well on other groups. ▶ A significant number of samples showed poor calculated and dialysis fT correlation. ▶ Commonly used fT equations may be too simplistic to encompass all patients.

Introduction

Measurement of serum testosterone concentrations is a central component of the assessment of androgen status. Increased concentrations can be seen in women with congenital adrenal hyperplasia or polycystic ovarian syndrome (PCOS), and in both genders due to exogenous testosterone administration, adrenal-, testicular-, or ovarian tumors, and in children with precocious puberty [1]. Conversely, decreased serum testosterone concentrations are the hallmark of both primary and secondary hypogonadism in men, and, to a lesser degree, women, regardless of the underlying etiology. Androgen deficiency in males is believed to be a particular common problem, affecting an estimated to 4.7 million men between the ages of 30 and 79 in the United States in 2000, and predicted to rise to 6.5 million by 2025 [2], [3]. Most of these cases represent mild to moderate deficiency, often referred to as “andropause” or “late-onset hypogonadism”, a state that is blamed for anergia, sexual dysfunction, declining mental ability, diminished muscle strength and reduced bone density [4], [5]. Decreased testosterone concentrations in women present with fewer and less specific symptoms [6], [7].

Measurement of total testosterone, often combined with gonadotropin measurements, is generally sufficient to diagnose significant androgen excess or deficiency. However, for suspected mild to moderate deficiency, measurement of the free, bioactive, fraction of testosterone (fT) is believed to be of superior diagnostic value, especially under circumstances of altered sex hormone binding globulin (SHBG) serum concentrations or binding affinity, as may be found in hyper- or hypothyroidism, liver cirrhosis, obesity, or exogenous sex hormone use, especially estrogen treatments [8], [9], [10], [11]. Serum concentrations of SHBG also increase with age, often affecting fT levels disproportionately to total testosterone concentrations [12].

Because the majority of testosterone is bound to SHBG or albumin, fT accounts for only 1–4% of the total testosterone levels. Furthermore, the protein-bound distribution of testosterone is gender dependent because of differences in sex steroid distribution, with 44% of testosterone bound to SHBG in men and 66% bound to SHBG in women [13]. These factors conspire to make measurements of fT challenging. The gold standard method of measurement is isotope dilution equilibrium dialysis [14], [15], [16]. While this method is considered the most accurate, its technical and laborious nature can result in high assay variability [17]. Analog immunoassays that detect fT have been proposed as an alternative. However, they have been widely criticized for their lack of accuracy and variability of results with fluctuating SHBG concentrations [18], [19], [20], suggesting that they do not truly measure fT.

Another alternative is to calculate fT using equations based on the law of mass action. These equations typically incorporate the results of serum measurements of total testosterone, albumin, and SHBG. The equation originally described by Sodergard et al. [21] and derived by Vermeulen et al. [22] also includes the testosterone association constants for albumin and SHBG. However, calculated results for fT have often been found to vary significantly from isotope dilution equilibrium dialysis. Potential sources for this variation include biological factors such inter-individual variability in the concentrations of competing hormones and binding proteins and in the affinity of testosterone for SHBG or albumin, and laboratory factors including different SHBG and testosterone methodologies, equations, and association constants used [23], [24], [25], [26]. Several different constants have been published and some studies have used empirically derived constants and equations to provide the best fit for their individual combinations of patient population, and total testosterone-, SHBG- and albumin assays [15], [27], [28], [29], [30], [31].

Overall, there appears to be little consensus on the best method for calculating fT, likely because of the wide variability of performance of these equations between studies, patient groups, and laboratories and a lack of understanding of how these factors impact calculated fT measurements. The purpose of this study was to improve our understanding of how these parameters affect calculated fT results, and to determine whether satisfactory agreement with isotope dilution equilibrium dialysis can be achieved. To this end we assessed the performance of three established equations and two new equations, generated using two different patient groups. These equations differed only in their testosterone association constants for SHBG and albumin. Results calculated with each of these five equations in these two patient groups were compared with those obtained by isotope dilution equilibrium dialysis performed on the same specimens. Demographic characteristics of the two patient populations and equation variables including albumin, SHBG, and choice of association constants were examined to determine how they affected the correlation with isotope dilution equilibrium dialysis results. Samples with poor correlation between calculated and measured fT results were examined further to find common characteristics that could explain the deviations.

Section snippets

Subjects

The Mayo Clinic Institutional Review Board approved this study. Non-fasting serum samples were obtained from two groups of patients that were seen for fT testing at Mayo Clinic Rochester. Group A consisted of 209 patients (171 males/38 females) seen between June and July, 2003. Group B consisted of 191 patients (93 males/98 females) seen between March and August, 2007. Group A consisted of all consecutive patients, while patients for Group B were intentionally selected to yield an equal number

Results

There was no significant impact of albumin concentration on calculated fT, regardless of whether the measured albumin results or a constant value of 43 g/L were used (y = 0.9733x + 0.0459, R2 = 0.99, data not shown), indicating that albumin concentrations seen in typical ambulatory patients have a negligible impact on the calculated fT concentration [22]. All subsequent calculations of fT were therefore made using the constant value of 43 g/L for albumin.

The agreement between calculated and isotope

Discussion

During the past decade, many laboratories have shifted away from isotope dilution equilibrium dialysis because of the cost and labor required and the amount of variability inherent in the assay. Measuring fT by ultrafiltration, while showing promising results in several studies [35], [36], [37], was found to have an unacceptable rate of device failure at our institution. The problems with these methods make calculated fT an attractive alternative. However, several publications have pointed out

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