CLINICAL RESEARCH ARTICLE

Plasma Free in States of Normal and Altered Binding : Implications for Adrenal Insufficiency Diagnosis

Laura E. Dichtel,1,2 Melanie Schorr,1,2 Claudia Loures de Assis,1 Elizabeth M. Rao,1 1 2,3 2,4 2,5

Jessica K. Sims, Kathleen E. Corey, Puja Kohli, Patrick M. Sluss, Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021 Michael J. McPhaul,6 and Karen K. Miller1,2

1Neuroendocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114; 2Harvard Medical School, Boston, Massachusetts 02114; 3Division of Gastroenterology, Massachusetts General Hospital, Boston, Massachusetts 02114; 4Division of Pulmonology and Critical Care Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114; 5Clinical Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114; and 6Endocrinology, Quest Diagnostics Nichols Institute, San Juan Capistrano, California 92675

ORCiD numbers: 0000-0001-6989-2308 (L. E. Dichtel).

Context: Accurate diagnosis of adrenal insufficiency is critical because there are risks associated with overdiagnosis and underdiagnosis. Data using liquid chromatography tandem mass spectrometry (LC/MS/MS) free cortisol (FC) assays in states of high or low cortisol-binding (CBG) levels, including cirrhosis, critical illness, and oral use, are needed.

Design: Cross-sectional.

Objective: Determine the relationship between CBG and as well as total cortisol (TC) and FC in states of normal and abnormal CBG. Establish the FC level by LC/MS/MS that best predicts TC of ,18 mg/dL (497 nmol/L) (standard adrenal insufficiency diagnostic cutoff) in healthy individuals.

Subjects: This study included a total of 338 subjects in four groups: healthy control (HC) subjects (n 5 243), patients with cirrhosis (n 5 38), intensive care unit patients (ICU) (n 5 26), and oral con- traceptive (OCP) users (n 5 31).

Main Outcome Measure(s): FC and TC by LC/MS/MS, albumin by spectrophotometry, and CBG by ELISA.

Results: TC correlated with FC in the ICU (R 5 0.91), HC (R 5 0.90), cirrhosis (R 5 0.86), and OCP (R 5 0.70) groups (all P , 0.0001). In receiver operator curve analysis in the HC group, FC of 0.9 mg/dL (24.8 nmol/L) predictedTCof,18 mg/dL (497 nmol/L; 98% sensitivity, 91% specificity; AUC, 0.98; P , 0.0001). Decreasing the cutoff to 0.7 mg/dL led to a small decrease in sensitivity (92%) with similar specificity (91%).

Conclusions: A cutoff FC of ,0.9 mg/dL (25 nmol/L) in this LC/MS/MS assay predicts TC of ,18 mg/dL (497 nmol/L) with excellent sensitivity and specificity. This FC cutoff may be helpful in ruling out adrenal insufficiency in patients with binding globulin derangements. (J Clin Endocrinol Metab 104: 4827–4836, 2019)

ccurate diagnosis of adrenal insufficiency is critical, (CBG) and albumin are the two main reservoirs of Aas there are serious risks of overdiagnosing and cortisol in the human body, binding 70% to 80% and underdiagnosing the condition. Cortisol-binding globulin 10% to 20% of total cortisol (TC), respectively.

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: CBG, cortisol-binding globulin; FC, free cortisol; HC, healthy control; ICU, Printed in USA intensive care unit; LC/MS/MS, liquid chromatography tandem mass spectrometry; OCP, Copyright © 2019 Endocrine Society oral contraceptive; TC, total cortisol. Received 4 January 2019. Accepted 16 April 2019. First Published Online 22 April 2019

doi: 10.1210/jc.2019-00022 J Clin Endocrinol Metab, October 2019, 104(10):4827–4836 https://academic.oup.com/jcem 4827 4828 Dichtel et al Plasma FC in States of Normal and Altered Binding Globulins J Clin Endocrinol Metab, October 2019, 104(10):4827–4836

Approximately 10% of TC is free or unbound, which the free cortisol level corresponding to the standard total reflects the biologically active cortisol in the body. States cortisol diagnostic cutoff of 18 mg/dL (497 nmol/L). of low CBG and albumin, such as critical illness and Having a robust FC cutoff for the diagnosis of adrenal cirrhosis, lead to underestimation of cortisol action by insufficiency would serve a great clinical need for patients TC levels, with the potential for overdiagnosis of adrenal with either low or high binding globulins because it insufficiency (1–5). In many instances, patients with would avoid exposing patients to the risks of infection, these diagnoses have complicated clinical courses, with bone loss, and adverse metabolic effects from steroid symptoms or signs that could be consistent with adrenal exposure, and would avoid putting them at risk of a insufficiency, such as hypotension, fatigue, and weak- missed diagnosis of true adrenal insufficiency. These ness, which make it particularly difficult to rule out in- clinical scenarios are encountered frequently at our in- Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021 sufficient cortisol production. On the other end of the stitution, which led to our examination of cortisol dynamics, spectrum, states of high CBG, such as oral contraceptive CBG, and albumin in subjects on oral contraceptives, with (OCP) use, lead to overestimation of cortisol action by cirrhosis, and in the intensive care unit (ICU). TC levels and the potential for underdiagnosis of adrenal Building upon this prior literature, we sought to use the insufficiency (6, 7). This is particularly relevant to women relationship between TC and FC in a large population with hypopituitarism on OCPs as treatment for central of healthy individuals with normal binding globulins to hypogonadism who may also need monitoring for the determine what FC corresponds to the standard TC cutoff development of adrenal insufficiency. used for the diagnosis of adrenal insufficiency (18 mg/dL Thus, although there are well-established cutoffs for or 497 nmol/L). We used LC/MS/MS assays for FC and the diagnosis of adrenal insufficiency for TC, the in- TC that are widely available for clinical care to maximize terpretation of measured levels is heavily dependent on the clinical utility and generalizability of our findings. We normal levels of binding globulins. In contrast, free additionally sought to determine whether albumin levels cortisol (FC) measurements more accurately reflect the could be used to predict CBG levels when interpreting TC free fraction of biologically active cortisol in the presence or whether there are clinical scenarios for which FC would of binding globulin derangements. However, normative be particularly useful. data for FC in the diagnosis of adrenal insufficiency is lacking. Moreover, establishment of normative serum FC Subjects and Methods values has been limited by prior use of older radioim- munoassays (8–11) or noncommercially available liquid Subjects chromatography tandem mass spectrometry (LC/MS/ Partners Human Research Committee (PHRC IRB) MS) assays, which have limited clinical application. approval was obtained for the use of discarded clinical Two recent small studies have proposed the use of FC for specimens with a waiver of informed consent from the diagnosis of adrenal insufficiency based on cosyn- subjects. Blood samples were obtained from discarded tropin stimulation data in subjects with adrenal in- clinical plasma specimens in a cross-sectional study sufficiency vs control subjects, with cutoffs ranging from across four subject groups, including HC subjects, OCP 0.9 to 1.3 mg/dL (25 to 36 nmol/L) by LC/MS/MS (12, users, individuals with cirrhosis, and ICU patients. A 13). One additional study examined a larger population total of 338 subjects in the four groups were studied: HC (n 5 295) that included all patients presenting to a single subjects (n 5 243), OCP users (n 5 31), patients with Endocrine Testing Center for evaluation of adrenal in- cirrhosis (n 5 38), and ICU patients (n 5 26). All subject sufficiency, with 14% of the cohort receiving the di- records were reviewed by a physician (L.E.D. or M.S.) for agnosis based on TC levels (14). Although their study medical history and medications prior to inclusion into was designed primarily to investigate cortisol dynamics the database and group assignment. Subjects across all on cosyntropin stimulation testing, the authors provided groups were excluded for systemic steroid use of any a FC cutoff of 0.9 and 1.2 mg/dL at 30 and 60 minutes, kind. Subjects with topical or inhaled steroid use were respectively. However, this study included women on included in the database (HC, n 5 11; ICU, n 5 3; oral estrogen as well as all patients attending the testing cirrhosis, n 5 2; and OCP, n 5 1). HC subjects were center who were not screened for conditions that could excluded for conditions that may affect binding globulin contribute to cortisol binding globulin or albumin de- levels such as cirrhosis, renal dysfunction, malignancy, rangements (14). OCP use, or human immunodeficiency virus. OCP users Thus, our study is the first to investigate the re- were subjected to the same exclusion criteria as HC lationship between total and free cortisol in a large subjects and were confirmed to be taking a combined population of healthy controls with normal cortisol estrogen- and -containing oral contracep- binding globulins in order to enable the determination of tive pill. ICU subjects were excluded if they had doi: 10.1210/jc.2019-00022 https://academic.oup.com/jcem 4829 concurrent cirrhosis, but all diagnoses were otherwise Scientific, Waltham, MA) and a tandem mass spec- included. Time of day and severity of illness varied based trometer (Thermo Scientific). Quantitation on the MS/MS on type of visit, which included outpatient and emer- is run in positive mode. The sensitivity of the method is gency room visits for the HC and OCP groups; out- 0.1 mg/dL. The interassay variation is ,8%: 1.5 mg/dL, patient, emergency room, and non-ICU admissions for CV 5 7.9%; 20 mg/dL, CV 5 6.8%; 40 mg/dL, CV 5 subjects with cirrhosis; and medical ICU admissions for 6.6%; and 80 mg/dL, CV 5 6.0%. The intra-assay var- all ICU subjects. The duration of ICU stay at the time of iation is 3.0% at 15.9 mg/dL and 4.6% at 202.7 mg/dL. blood draw was 3.0 6 2.2 (SD), and mean total duration Free cortisol was measured after equilibrium dialysis as of ICU stay was 5.5 6 4.4 (SD) days. Model for End- previously described (12). Samples were incubated in a Stage Disease scores and Sequential Organ Failure dialysis chamber against PBS buffer at 37°C. After Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021 Assessment scores were calculated as a measure of disease reaching equilibrium, an aliquot from the PBS chamber FC severity for individuals in the cirrhosis and ICU groups, was analyzed using the LC/MS/MS method described respectively. Mean Model for End-Stage Liver Disease above. The sensitivity of the serum FC method is 0.03 mg/ score of the cirrhosis group was 14 6 7 (subject score dL. The FC interassay variation was ,10% (9.4% at range, 6 to 30; possible score range, 6 to 40), which 0.36 mg/dL and 9.8% at 2.17 mg/dL). The FC intra-assay represents ;6% 3-month mortality (15). Mean Se- variation was 7.4% at 0.36 mg/dL and 9.3% at quential Organ Failure Assessment score on admission 2.20 mg/dL. for the ICU group was 6.2 6 3.1 (subject score range, 2 Albumin was measured spectrophotometrically on the to 14; possible score range, 0 to 24), which repre- Beckman Coulter AU series platform (Brea, CA) using a sents ,10% hospital mortality (16, 17). Demographics dye-binding method (Albumin, Beckman Coulter, Brea, and baseline laboratory values are listed in Table 1. CA and Quest Diagnostics Nichols Institute, San Juan Capistrano, CA). At acid pH, bromocresol green reacts Methods with albumin to form an intense green complex, and the FC and TC were measured by an isotope dilution mass formation of the color of this complex is measured spectrophotometric LC/MS/MS method as previously bichromatically (600/800 nm). The intensity is directly described (12) (Quest Diagnostics Nichols Institute, San proportional to the albumin concentration in the sample. Juan Capistrano, CA). Briefly, TC and FC were measured The sensitivity of the method is 1.5 g/dL (interassay using an on-line extraction method involving a high- variation ,1.0%: 2.1 g/dL, 0.5%; 4.5 g/dL, 0.4%; 5.3 g/dL, throughput liquid chromatography system (Thermo 0.4%; normal range 3.6 to 5.1 g/dL).

Table 1. Subject Characteristics and Laboratory Values

HC Subjects (n 5 243) OCP Users (n 5 31) Cirrhosis (n 5 38) ICU (n 5 26) Subject demographics Age, y 47 6 16A 30 6 8B 56 6 12C 60 6 16C Female, n (%) 130 (53) 31 (100) 12 (32) 12 (46) BMI, kg/m2 27.9 6 5.9AB 24.9 6 3.6B 29.6 6 8.3A 30.2 6 10.5A White, n (%) 170 (70) 25 (81) 28 (74) 21 (81) Outpatient, n (%) 141 (58) 27 (87) 24 (63) N/A ER visit, n (%) 102 (42) 4 (13) 0 (0) N/A Inpatient, n (%) N/A N/A 14 (37) 26 (100) Laboratory valuesa Total , g/dL 7.1 6 0.3A 6.8 6 0.4AB 6.8 6 0.9A 5.9 6 0.9B Total bili, mg/dL 0.6 6 0.8A 0.3 6 0.1A 4.3 6 7.1B 0.7 6 0.8A AST, U/L 32 6 4A 24 6 13A 81 6 63B 61 6 61B ALT, U/L 30 6 57A 19 6 10A 43 6 33B 52 6 54AB Sodium, mmol/L 141 6 3AB 142 6 3AB 138 6 7B 140 6 4A Creatinine, mg/dL 0.86 6 0.20AB 0.77 6 0.20AB 0.83 6 0.70A 1.27 6 1.10B INR 1.3 6 0.3 1.2 6 0.3 Disease-specific risk scores MELD score 14 6 7 SOFA score 6.2 6 3.1

Variables not referenced by the same letter indicate a significant difference between groups (P , 0.05). Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; ER, emergency room; INR, international nor- malized ratio; MELD, Model for End-Stage Liver Disease; N/A, not available; SOFA, Sequential Organ Failure Assessment. aSubset of HC subjects and OCP users defined as follows. Total protein: HC n 5 38, OCP n 5 17; total bilirubin: HC n 5 134, OCP n 5 10; AST and ALT: HC n 5 139, OCP n512; Na: HC n 5 214, OCP n 5 21; Cr: HC n 5 212, OCP n 5 21. 4830 Dichtel et al Plasma FC in States of Normal and Altered Binding Globulins J Clin Endocrinol Metab, October 2019, 104(10):4827–4836

CBG was measured at the Research Laboratory Ser- Results vices at Maine Medical Center using a sandwich ELISA with an intra-assay variation of 1.7% and interassay Determination of FC cutoff variation of 7.1% (Biovendor, Ashville, NC). In receiver operator curve analysis in the HC group, an FC cutoff of 0.9 mg/dL (25 nmol/L) predicted a diagnosis Statistical analysis of adrenal insufficiency as defined as a TC of ,18 mg/mL Analysis was conducted using JMP Statistical Dis- with 98% sensitivity (3/185 false positives) and 89% covery Software, Version 13 Professional (SAS Institute, specificity (3/35 false negatives; AUC, 0.98; P , 0.0001) Inc., Cary, NC). Variables were log-transformed prior to (Fig. 1). The sensitivity (i.e., the probability of a true analysis. Four group comparisons were performed using diagnosis to rule in adrenal insufficiency) was 100% at a Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021 the Tukey-Kramer multiple comparisons test. Linear cutoff of 1.5 mg/dL (41 nmol/L), remained robust at the regression analysis with Pearson correlation coefficients proposed cutoff of 0.9 mg/dL (25 nmol/L, 98%), and was used to investigate the associations between vari- began dropping below 0.7 mg/dL (19 nmol/L, 92%). The ables. A two-tailed P value #0.05 defined statistical specificity (i.e., the probability of a true negative result to significance. Unless otherwise specified, data are reported rule out adrenal insufficiency) remained stable at 91% as mean 6 SD or as an R coefficient and P value. Slopes down to a cutoff of 0.7 mg/dL (19 nmol/L) and only rose were compared with Dunnett test with HC subjects as the to 97% at a cutoff of 0.4 mg/dL (11 nmol/L). Six in- reference group. Receiver operator curve analysis was dividuals had discordant results on the two cutoffs performed to calculate the FC with greatest sensitivity of TC ,18 mg/dL (497 nmol/L) and FC ,0.9 mg/dL and specificity for the diagnosis of adrenal insufficiency. (25 nmol/L) for the diagnosis of adrenal insufficiency. Adrenal sufficiency was defined as the negative condition Three HC subjects had a TC .18 mg/dL (497 nmol/L) and adrenal insufficiency as the positive condition using but had FC ,0.9 mg/dL (25 nmol/L), including TC the standard TC cutoffs of $18 and ,18 mg/dL, 18.3 mg/dL (505 nmol/L) and FC 0.4 mg/dL (11 nmol/L), respectively. TC 18.8 mg/dL (519 nmol/L) and FC 0.6 mg/dL (17 nmol/L),

Figure 1. Sensitivity (triangles and dotted line) and specificity (squares and solid line) of different plasma FC cutoffs for the diagnosis of adrenal insufficiency. True positive, true negative, false positive, and false negative data for each FC cutoff are noted. doi: 10.1210/jc.2019-00022 https://academic.oup.com/jcem 4831 and TC 21.6 mg/dL (596 nmol/L) and FC 0.7 mg/dL (19 samples and severity of illness precludes meaningful nmol/L). Similarly, three HC subjects had a TC ,18 mg/dL comparison of absolute levels between groups. As ex- (497 nmol/L) and FC .0.9 mg/dL (25 nmol/L), including pected, percent FC was significantly higher in the ICU TC 17.1 mg/dL (472 nmol/L) and FC 1.2 mg/dL (33 nmol/ subjects (7.7 6 4.8%), a group with a high illness severity L), TC 17.2 mg/dL (475 nmol/L) and FC 1.1 mg/dL and low CBG levels, compared with the HC (4.2 6 2.2%, (30 nmol/L), and TC 17.7 mg/dL (488 nmol/L) and FC P , 0.0001), cirrhosis (5.4 6 2.7%, P , 0.006), and OCP 1.5 mg/dL (41 nmol/L). Thus, the latter three HC subjects groups (1.8 6 0.8%, P , 0.0001). Percent FC was sig- deemed adrenally sufficient by the FC assay had near- nificantly lower in the OCP group, a predominantly passing TC levels (all .17.0 mg/dL or 469 nmol/L). The FC outpatient sample with high CBG levels, when compared cutoff as well as its sensitivity and specificity for the di- with all other groups (P , 0.0001 for all comparisons) Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021 agnosis of adrenal insufficiency were unchanged after the (Table 2). ICU subjects with albumin ,2.5 g/dL had exclusion of the HC subjects who were taking inhaled higher FC despite similar TC as those subjects with an steroids (n 5 11). albumin of $2.5 g/dL (FC 1.8 6 1.2 vs 0.6 6 0.7 mg/dL, P , 0.0005; TC 13.6 6 5.5 vs 12.1 6 7.6 mg/dL, P 5 0.2). Albumin and CBG TC strongly correlated with FC in the HC (R 5 0.90, Mean albumin in the OCP group did not differ from P , 0.0001), ICU (R 5 0.91, P , 0.0001), and cirrhosis that of the HC group (4.6 6 0.3 vs 4.7 6 0.3 g/dL, re- (R 5 0.86, P , 0.0001) groups but more weakly in the spectively; P 5 NS). However, both the cirrhosis (3.5 6 OCP group (R 5 0.70, P , 0.0001) (Fig. 4A–4D). These 0.9 g/dL) and ICU (3.1 6 0.7 g/dL) groups had lower mean four TC and FC correlation plots are shown on the same albumin levels than the HC group (both comparisons, P , axis in Fig. 4E for direct comparison. 0.0001) (Table 2; Fig. 2A). As expected, mean CBG was 6 m higher in the OCP (57 18 g/mL) group and lower in the Discussion ICU (22 6 4 mg/mL) and cirrhosis (21 6 7 mg/mL) groups compared with the HC group (28 6 7 mg/mL, P , 0.0001 We investigated albumin, CBG, TC, and FC levels for all other groups vs HC) (Table 2; Fig. 2B). across four populations to better care for patients with CBG correlated weakly with albumin in the HC (R 5 alterations in cortisol binding . Specifically, we 0.32, P , 0.0001) (Fig. 3A) and cirrhosis groups (R 5 identified an FC level that corresponds to a TC level of 0.46, P 5 0.01) (Fig. 3B) but was not correlated in the OCP 18 mg/dL (497 nmol/L) in HC subjects with normal (R 5 0.17, P 5 0.4) or ICU groups (R 5 0.19, P 5 0.4) binding globulins to help establish an FC cutoff for the (Fig. 3C and 3D). There was a trend toward a higher CBG diagnosis of adrenal insufficiency. Our hope is that the in the women on higher-dose ($0.03 mg estra- development of a standardized FC cutoff for the di- diol; n 5 20; CBG, 62 6 19 mg/mL) vs lower dose estradiol agnosis of adrenal insufficiency could greatly affect (#0.02 mg estradiol; n 5 9; CBG, 48 6 13 mg/mL; P 5 patient care because it could be applied to patients with 0.08). However, there was notable within-group vari- alterations in cortisol binding globulins who may oth- ability and overlap in CBG levels between the high-dose erwise be over- or underdiagnosed with adrenal in- and low-dose groups (range, 31 to 100 mg/mL vs 26 to sufficiency by TC levels alone. With this in mind, we 62 mg/mL, respectively). also investigated the ability to use albumin as a proxy for CBG as well as the relationship between TC and FC TC and FC to identify specific patient populations in whom TC may Absolute TC and FC by group are presented in be particularly misleading and require the use of a FC Table 2; however, variability in timing of unstimulated cutoff.

Table 2. Cortisol and Binding Globulin Results

HC Subjects (n 5 243) OCP Users (n 5 31) Cirrhosis (n 5 38) ICU (n 5 26) Binding globulin variables Albumin, g/dL 4.7 6 0.3A 4.6 6 0.3A 3.5 6 0.9B 3.1 6 0.7C CBG, mg/mL 28 6 7A 57 6 18B 21 6 7C 22 6 4C Cortisol variables TC, mg/dL 11.5 6 7.3A 18.9 6 8.5B 9.9 6 5.7A 14.7 6 6.6AB FC, mg/dL 0.58 6 0.67A 0.33 6 0.15A 0.59 6 0.47A 1.29 6 1.08B FC, % 4.2 6 2.2A 1.8 6 0.8B 5.4 6 2.7A 7.7 6 4.8C

Variables not referenced by the same letter indicate a significant difference between groups (P , 0.05). Multiply by 27.6 to convert cortisol from mg/dL to nmol/L. Multiply by 17.2 to convert CBG from mg/mL to nmol/L. Multiply by 10 to convert albumin from g/dL to g/L. 4832 Dichtel et al Plasma FC in States of Normal and Altered Binding Globulins J Clin Endocrinol Metab, October 2019, 104(10):4827–4836 Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021

Figure 2. (A) Albumin and (B) CBG by study group. *P , 0.01; ***P , 0.0001.

Thus, our primary aim was to establish an FC level women on oral estrogen). A study of patients with that corresponds to the gold-standard TC cutoff for the adrenally insufficiency (n 5 10) and control subjects (n 5 12) diagnosis of adrenal insufficiency in healthy individuals proposed a cutoff of 0.9 mg/mL at 30 minutes and 1.3 mg/mL (18 mg/dL or 497 nmol/L). Three prior studies defining at 60 minutes in this same assay as the current study (Quest FC cutoffs for the diagnosis of adrenal insufficiency are Diagnostics) after a 250-mg standard dose cosyntropin worth reviewing. Bancos et al. (14) studied a larger stimulation test (12). Rauschecker et al. (13) also obtained an number of individuals (n 5 295) with unknown adrenal FC cutoff of 0.9 mg/mL in the same LC/MS/MS assay as our status and determined separate FC cutoffs for 30- and 60- study (Quest Diagnostics) using cosyntropin-stimulated minute values (0.87 and 1.17 mg/mL in all subjects, re- values in patients with adrenal insufficiency (n 5 43) vs spectively, and 0.90 and 1.20 mg/mL when excluding control subjects (n 5 27).

Figure 3. Correlation between albumin and CBG by study group, including (A) HC, (B) cirrhosis, (C) ICU, and (D) OCP. Logged correlations shown with R and P values reported on individual graphs. doi: 10.1210/jc.2019-00022 https://academic.oup.com/jcem 4833 Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021

Figure 4. (A–D) Correlation between FC and TC by individual study group and (E) plotted by group on the same axis for comparison. Unlogged correlations shown with R and P values reported for analysis after log transformation.

Our data are consistent with these studies in that they cutoff indicates that it is unlikely to miss a critical di- demonstrate that a plasma FC cutoff of ,0.9 mg/mL agnosis of adrenal insufficiency (i.e., negative results are (25 nmol/L) predicts a TC of ,18 mg/dL (497 nmol/L) true negatives). In addition, our data suggest that the in a large group of healthy individuals with normal sensitivity for the diagnosis of adrenal insufficiency binding globulins with excellent sensitivity and good remained high (92%) when the cutoff was decreased to specificity. This cutoff was reached using a larger pop- 0.7 mg/dL (19 nmol/L), indicating that a lower cutoff may ulation and a different methodology than prior studies, still diagnose the majority of cases of adrenal insufficiency which we believe adds to the literature in fortifying and while reliably excluding the diagnosis in most patients. establishing the utility of this FC cutoff in clinical care. Additionally, our data demonstrate that, although This could be a useful tool in clinical practice for patients albumin and CBG significantly correlated with each with altered binding globulins in whom TC may be un- other in healthy individuals and patients with cirrhosis, reliable, particularly because the high sensitivity of the albumin was a poor proxy for CBG overall, particularly 4834 Dichtel et al Plasma FC in States of Normal and Altered Binding Globulins J Clin Endocrinol Metab, October 2019, 104(10):4827–4836 in critically ill patients in the ICU and healthy women on However, they are limited by variable testing protocols, OCPs. Thus, hypoalbuminemia in a patient with cir- the use of immunoassays, and the lack of design to rhosis may truly reflect a reduction in both albumin and specifically determine serum FC cutoffs for this purpose CBG, which would significantly alter the interpretation (18, 19, 23). of TC levels in these patients. However, these data The physiology of changes in albumin, CBG, TC, and suggest that, although albumin was shown to be useful FC levels in states of critical illness are complex and as a dichotomous predictor of the accuracy of TC in the difficult to study in a heterogenous ICU population. ICU (1), it is not helpful as a continuous predictor of CBG Studies in the ICU population have demonstrated dif- levels in critical illness. These discrepant findings have ferential changes in albumin and CBG (19) and changes been reported elsewhere and may be due to the hetero- in binding dynamics as TC increases (24) and suggest that Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021 geneity of ICU populations, which often include patients the proportion of FC to TC may be different in these with septic shock, sepsis, and nonsepsis-related shock patients irrespective of binding globulins (18). Thus, and critical illness (18–20). prospective studies should be performed in critically ill Moreover, healthy women on OCPs had a very var- patients to further investigate the physiology of TC, FC, iable, and at times robust, elevation of CBG, which and binding globulins in this setting. Additionally, some resulted in a poor correlation with albumin levels. Oral authors suggest that critically ill patients would be ex- estrogen is known to increase CBG levels (21), with a pected to have an FC that potentially surpasses a healthy, resulting increase in TC levels (6, 22). Overall, our data stimulated individual (20), suggesting that the same FC support the notion that higher estrogen doses in OCPs cutoff defined in this paper may not be applicable to the lead to higher CBG levels. However, there was still ICU population in particular. substantial variability of CBG levels even among those on We recognize the difficulty of translating plasma FC high or low estradiol doses, which limits the ability to and TC levels to physiologic endpoints due to the predict how any one individual’s CBG level will respond complexity of this steroid and binding globulin system. to OCP administration in the clinical setting. Thus, our Cortisol exhibits diurnal variability and wide fluctua- data also strongly suggest that TC should not be used to tions based on severity of illness, resulting in expected assess the adrenal axis in oral contraceptive users because normal ranges that are condition dependent. We addi- it could overestimate a patient’s FC status, particularly in tionally recognize the limitation of a single timepoint the unstimulated state. In our own neuroendocrine measurement of a free hormone, which is in constant practice (L.E.D., K.K.M., and M.S.), we frequently en- equilibrium that can be affected by a wide variety of counter women on OCPs who are at risk for adrenal factors, including inflammation, temperature, and other insufficiency and need axis testing. Because the effect on rapidly changing conditions. Moreover, the cellular re- CBG is long lasting, OCPs must be stopped for months to sponse to cortisol is durable, which stands in stark allow for accurate TC testing, delaying the diagnosis of contrast to the rapid fluctuations of plasma cortisol adrenal insufficiency and leaving these women at risk for levels. Finally, CBG is a subject to degradation by potential unintended pregnancy as well as menstrual, enzymes such as neutrophil elastace, which provides mood, and other unintended effects of OCP withdrawal. targeted delivery of liberated FC to local tissues in the Therefore, this is an ideal patient population for which inflammatory state (25). Thus, the measurement of TC utilization of FC testing for adrenal insufficiency makes and FC levels may not adequately reflect the underlying an important clinical impact. complexity of this steroid system and the resulting The ICU population deserves particular attention due physiologic response. to the complex physiology of cortisol and binding Limitations of our study include a lack of cosyntropin globulins in these patients, and there is a body of liter- stimulation testing across the four main study groups. ature regarding TC and FC in this group. A seminal paper Although random sampling allowed us to obtain a wide by Hamrahian et al. (1) demonstrated that serum TC range of TC and FC levels, it limited our ability to underestimated serum FC (measured by radioimmuno- compare TC and FC levels across our subject groups. assay) in critically ill patients with hypoalbuminemia and Additionally, although still widely used in clinical care, that measurement of TC in this population would lead to our gold standard TC cutoff of 18 mg/dL (497 nmol/L) overdiagnosis of adrenal insufficiency. However, this was established using older, nonspecific fluorometric study was performed using an immunoassay and was not assays, which might not reflect the same levels measured designed to determine an FC cutoff for the diagnosis of by current LC/MS assays or even radioimmunoassays adrenal insufficiency (1). There are additional studies (8–11, 26–28), although one recent study effectively investigating the use of serum or salivary FC in the di- confirmed these cutoffs in an LC/MS/MS assay (26). agnosis of adrenal insufficiency in the ICU population. Finally, we were limited in our ability to fully evaluate the doi: 10.1210/jc.2019-00022 https://academic.oup.com/jcem 4835 complex nature of albumin, CBG, and cortisol binding 5. Thevenot T, Borot S, Remy-Martin A, Sapin R, Cervoni JP, Richou dynamics in our heterogeneous ICU population as noted C, Vanlemmens C, Cleau D, Muel E, Minello A, Tirziu S, Penfornis A, Di Martino V, Monnet E. Assessment of adrenal function in above. This may limit the clinical application of our FC cirrhotic patients using concentration of serum-free and salivary cutoff in critical illness. cortisol. Liver Int. 2011;31(3):425–433. In summary, an FC of ,0.9 mg/dL (25 nmol/L) in this 6. Jung C, Ho JT, Torpy DJ, Rogers A, Doogue M, Lewis JG, Czajko RJ, Inder WJ. A longitudinal study of plasma and urinary cortisol in commercially available LC/MS/MS assay predicts a di- pregnancy and postpartum. J Clin Endocrinol Metab. 2011;96(5): agnosis of adrenal insufficiency with maximal sensitiv- 1533–1540. ity and specificity based on the standard TC cutoff 7. Qureshi AC, Bahri A, Breen LA, Barnes SC, Powrie JK, Thomas of ,18 mg/dL (497 nmol/L) in healthy individuals. Thus, SM, Carroll PV. The influence of the route of oestrogen admin- istration on serum levels of cortisol-binding globulin and total an FC level of ,0.9 mg/dL (25 nmol/L) could be used in 66 – cortisol. Clin Endocrinol (Oxf). 2007; (5):632 635. Downloaded from https://academic.oup.com/jcem/article/104/10/4827/5475551 by guest on 26 September 2021 clinical care to aid in the diagnosis of adrenal in- 8. El-Farhan N, Rees DA, Evans C. Measuring cortisol in serum, urine sufficiency in states of altered CBG, particularly in those and saliva: are our assays good enough? Ann Clin Biochem. 2017; 54(3):308–322. with cirrhosis or on oral estrogen, with the caveat that the 9. El-Farhan N, Pickett A, Ducroq D, Bailey C, Mitchem K, Morgan sensitivity and specificity did not reach 100% even in HC N, Armston A, Jones L, Evans C, Rees DA. Method-specific serum subjects, and clinical judgment must be used in inter- cortisol responses to the adrenocorticotrophin test: comparison of pretation of TC and FC levels in specific clinical cases. gas chromatography-mass spectrometry and five automated im- munoassays. Clin Endocrinol (Oxf). 2013;78(5):673–680. Certain groups, such as those with critical illness in the 10. Rosner W, Vesper H. Preface. CDC workshop report improving ICU, deserve further study given the complexity of measurements in patient care and research cortisol system dynamics in this population. translation. Steroids. 2008;73(13):1285. 11. Burt MG, Mangelsdorf BL, Rogers A, Burt MG, Mangelsdorf BL, Rogers A, Ho JT, Lewis JG, Inder WJ, Doogue MP. Free and total Acknowledgments plasma cortisol measured by immunoassay and mass spectrometry following ACTH1–24 stimulation in the assessment of pituitary 98 – The authors dedicate this manuscript to the late Dr. Richard patients. J Clin Endocrinol Metab. 2013; (5):1883 1890. 12. Peechakara S, Bena J, Clarke NJ, McPhaul MJ, Reitz RE, Weil RJ, Reitz, who made a substantial contribution to this work before Recinos P, Kennedy L, Hamrahian AH. Total and free cortisol his passing. levels during 1 mg, 25 mg, and 250 mg cosyntropin stimulation tests Financial Support: This work was supported by the fol- compared to insulin tolerance test: results of a randomized, pro- lowing National Institutes of Health Grants: K23DK113220 (to spective, pilot study. Endocrine. 2017;57(3):388–393. L.E.D.), K23DK115903 (to M.S.), and K24HL092902 (to 13. Rauschecker M, Abraham SB, Abel BS, Wesley R, Saverino E, K.K.M.). Trivedi A, Heller T, Nieman LK. Cosyntropin-stimulated serum Correspondence and Reprint Requests: free cortisol in healthy, adrenally insufficient, and mildly cir- Laura E. Dichtel, rhotic populations. J Clin Endocrinol Metab. 2016;101(3): MD, MHS, Neuroendocrine Unit, Massachusetts General 1075–1081. Hospital, BUL457B, 55 Fruit Street, Boston, Massachusetts 14. Bancos I, Erickson D, Bryant S, Hines J, Nippoldt TB, Natt N, 02114. E-mail: [email protected]. Singh R. Performance of free versus total cortisol following Disclosure Summary: Laboratory services were provided cosyntropin stimulation testing in an outpatient setting. Endocr Pract. 2015;21(12):1353–1363. at no cost by Quest Diagnostics, San Juan Capistrano, CA. 15. Kamath PS, Kim WR; Advanced Liver Disease Study Group. The M.J.M. is an employee of Quest Diagnostics, San Juan Cap- model for end-stage liver disease (MELD). Hepatology. 2007; istrano, CA. Discounted Cortisol Binding Globulin kits were 45(3):797–805. provided by Biovendor, Ashville, NC. 16. 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Molenaar N, Johan Groeneveld AB, Dijstelbloem HM, de Jong different information regarding the hypothalamus-pituitary- MF, Girbes AR, Heijboer AC, Beishuizen A. Assessing adrenal adrenal axis reserve in patients with liver impairment. Ann Clin insufficiency of secretion using free versus total Biochem. 2009;46(Pt 6):505–507. cortisol levels in critical illness. Intensive Care Med. 2011;37(12): 3. Tan T, Chang L, Woodward A, McWhinney B, Galligan J, 1986–1993. Macdonald GA, Cohen J, Venkatesh B. Characterising adrenal 19. Ho JT, Al-Musalhi H, Chapman MJ, Quach T, Thomas PD, Bagley function using directly measured plasma free cortisol in stable CJ, Lewis JG, Torpy DJ. Septic shock and sepsis: a comparison of severe liver disease. J Hepatol. 2010;53(5):841–848. total and free plasma cortisol levels. J Clin Endocrinol Metab. 4. Galbois A, Rudler M, Massard J, Fulla Y, Bennani A, Bonnefont- 2006;91(1):105–114. Rousselot D, Thibault V, Reignier S, Bourrier A, Poynard T, 20. 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