Handbook of Diagnostic Endocrinology - part 1 pptx

In Handbook of Diagnostic Endocrinology we provide the reader with a concise approach to the diagnosis of endocrine disorders that is based on anunderstanding of their pathophysiology an

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Handbook of

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1 Endocrine glands—Diseases—Diagnosis—Handbooks, manuals, etc I Hall, Janet E.

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P REFACE

The aim of the Handbook of Diagnostic Endocrinology is to provide a

com-prehensive overview of current approaches to the diagnosis of endocrine ders Our ability to diagnose patients with diseases of the endocrine systems isexpanding exponentially with the development of new and more reliable assaymethods and the incorporation of both molecular and genetic approaches intoour understanding of the pathophysiology of these diseases Although the pri-mary focus of this volume is on the diagnosis of endocrine disease, the vastmajority of endocrine diseases require long-term management; therefore, many

disor-of the chapters also discuss approaches to follow-up in these patients

The Handbook of Diagnostic Endocrinology comprises 16 chapters

Immu-noassays have long been the cornerstones of endocrine diagnoses As an tant background, Sabrina Gill, Frances Hayes, and Patrick Sluss discuss themany methodological advances that have expanded our repertoire of diagnostictests With the increased number of tests available for endocrine evaluation, it

impor-is incumbent that we understand the factors that affect assay performance andtheir impact on our ability to use these tools to aid in clinical diagnosis JosephVerbalis reviews the regulatory mechanisms underlying water and sodiummetabolism and presents a comprehensive approach to disorders of body fluids,which are among the most commonly encountered problems in clinical medi-cine Mary Lee Vance outlines the diagnosis and long-term followup of patientspresenting with pituitary tumors, and Lynnette Nieman provides an approach tothe diagnosis and differential diagnosis of Cushing’s syndrome that includes acritical appraisal of available tests Although endocrine causes of hypertensionoccur in only 10% of hypertensive subjects, this small fraction represents a largenumber of patients Jennifer Lawrence and Robert Dluhy present a thoughtfuland efficient approach to the diagnosis of these disorders Anastassios Pittas andStephanie Lee then update the approach to the diagnosis of thyroid disease,including an important discussion of currently available assays Regina Castroand Hossein Gharib present a cost-effective approach to evaluation of the com-mon problem of thyroid nodules and discuss the preoperative evaluation andpostoperative followup of patients with thyroid cancer Allison Goldfine pro-vides an overview of the diagnosis of the various forms of diabetes and a com-prehensive discussion of long-term monitoring of the primary disease and itscomplications Robert Ratner reviews the important area of gestational diabetes,both its diagnosis and consequences, and John Service presents a clear approach

to the diagnosis of hypoglycemia William Donahoo, Elizabeth Stephens, and

vi Preface

Robert Eckel provide a thorough discussion of the modalities that are currentlyavailable for the assessment of dyslipidemia and obesity Benjamin Leder andJoel Finkelstein then review calcium metabolism and present a logical approach

to the diagnosis of hyper- and hypocalcemia Patrick Doran and Sundeep Khosladiscuss the spectrum of osteoporosis and an approach to diagnosis and long-termfollowup based on the currently available tools Margaret Wierman reviews thenormal physiology of the hypothalamic–pituitary–testicular axis and the physi-ology of erection, both of which are critical to the approach to evaluation ofhypogonadism and erectile dysfunction Drew Tortoriello and Janet Hall thendiscuss the physiology of normal menstrual function as a backdrop to theirapproach to the evaluation and long-term followup of women with disorders ofmenstrual function Ricardo Azziz completes this volume with a practical andfocused discussion of the evaluation of androgen excess in women

In Handbook of Diagnostic Endocrinology we provide the reader with a

concise approach to the diagnosis of endocrine disorders that is based on anunderstanding of their pathophysiology and includes both clinical manifesta-tions and the most current laboratory tests available This work will serve as areference for students and fellows in training as well as an update for practicingendocrinologists and internists

We thank the contributors to this volume, without whose expertise and effortsthis work would not be possible

Janet E Hall, MD Lynnette K Nieman, MD

C ONTENTS

Preface vContributors ix

1 Issues in Endocrine Immunoassay 1

Sabrina Gill, Frances J Hayes, and Patrick M Sluss

2 Disorders of Water Metabolism 23

Joseph G Verbalis

3 Pituitary Tumors: Prolactinomas, Acromegaly,

Gonadotropin-Producing, Nonfunctioning 55 Mary Lee Vance

4 Cushing’s Syndrome 67

Lynnette K Nieman

5 Endocrine Hypertension 85

Jennifer E Lawrence and Robert G Dluhy

6 Evaluation of Thyroid Function 107

Anastassios G Pittas and Stephanie L Lee

7 Thyroid Nodules and Thyroid Cancer 131

M Regina Castro and Hossein Gharib

8 Diagnosis and Management of Diabetes 157

Allison B Goldfine

9 Gestational Diabetes: Where Do We Look for It

and How Do We Find It? 179 Robert E Ratner

10 Hypoglycemic Disorders 193

F John Service

11 The Evaluation of Dyslipidemia and Obesity 213

William T Donahoo, Elizabeth Stephens, and Robert H Eckel

12 Hyper- and Hypocalcemia 239

Benjamin Z Leder and Joel S Finkelstein

viii Contents

13 Osteoporosis 257

Patrick M Doran and Sundeep Khosla

14 Hypogonadism and Erectile Dysfunction 277

Margaret E Wierman

15 Menstrual Dysfunction 295

Drew V Tortoriello and Janet E Hall

16 Differential Diagnosis and Evaluation

of Hyperandrogenism 323

Ricardo Azziz

Index 331

C ONTRIBUTORS

RICARDO AZZIZ, MD, MPH, MBA, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA

M REGINA CASTRO, MD, Department of Medicine and Endocrinology, Albany

Medical College, Stratton VA Medical Center, Albany, NY

ROBERT G DLUHY, MD, Division of Endocrinology-Hypertension, Brigham

and Women’s Hospital, Boston, MA

WILLIAM T DONAHOO, MD, Division of Endocrinology, Metabolism and

Diabetes, University of Colorado Health Sciences Center, Denver, CO

PATRICK M DORAN, MD, Division of Endocrinology, McGill University Health

Center, Montreal, Canada

ROBERT H ECKEL, MD, Division of Endocrinology, Metabolism and Diabetes,

University of Colorado Health Sciences Center, Denver, CO

JOEL S FINKELSTEIN, MD, Division of Endocrinology, Massachusetts General

Hospital, Boston, MA

HOSSEIN GHARIB, MD, FACE, Division of Endocrinology and Metabolism, Mayo

Clinic and Mayo Foundation, Rochester, MN

SABRINA GILL, MD, Division of Endocrinology and Metabolism, St Paul’s

Hospital, Vancouver BC, Canada

ALLISON B GOLDFINE, MD, Research Division, Joslin Diabetes Center, Boston,

MA

JANET E HALL, MD, Reproductive Endocrine Unit, Division of Endocrinology,

Massachusetts General Hospital, Boston, MA

FRANCES J HAYES, MD, Reproductive Endocrine Unit, Division of

Endocrinology, Massachusetts General Hospital, Boston, MA

SUNDEEP KHOSLA, MD, Division of Endocrinology and Metabolism, Mayo

Clinic, Rochester, MN

JENNIFER E LAWRENCE, MD, Valdosta Specialty Clinic, Valdosta, GA

BENJAMIN Z LEDER, MD, Division of Endocrinology, Massachusetts General

Hospital, Boston, MA

STEPHANIE L LEE, MD, Division of Endocrinology, Diabetes and Metabolism,

Boston Medical Center, Boston, MA

LYNNETTE K NIEMAN, MD, Pediatric and Reproductive Endocrinology Branch,

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD

ANASTASSIOS G PITTAS, MD, Division of Endocrinology, Diabetes, Metabolism

and Molecular Medicine, New England Medical Center, Boston, MA

ROBERT E RATNER, MD, MedStar Research Institute, Washington, DC

F JOHN SERVICE, MD, P D, Division of Endocrinology and Internal Medicine,

Mayo Clinic, Rochester, MN

PATRICK M SLUSS, P D, Reproductive Endocrine Unit, Division of

Endocrinology, Massachusetts General Hospital, Boston, MA

ELIZABETH STEPHENS, MD, Division of Endocrinology, Diabetes, and Clinical

Nutrition, Oregon Health and Science University, Portland, OR

DREW V TORTORIELLO, MD, Department of Obstetrics and Gynecology,

Columbia Presbyterian Medical Center, New York, NY

MARY LEE VANCE, MD, Division of Endocrinology, University of Virginia

Health Services Center, Charlottesville, VA

JOSEPH G VERBALIS, MD, Division of Endocrinology and Metabolism,

Georgetown University School of Medicine, Washington, DC

MARGARET E WIERMAN, MD, Division of Endocrinology, Veterans Affairs Medical Center, University of Colorado School of Medicine, Denver, CO

Chapter 1/Endocrine Immunoassay 1

From: Contemporary Endocrinology: Handbook of Diagnostic Endocrinology

Edited by: J E Hall and L K Nieman © Humana Press Inc., Totowa, NJ

1 Issues in Endocrine Immunoassay

Sabrina Gill, MD , Frances J Hayes, MD , and Patrick M Sluss, P h D

CONTENTS

INTRODUCTION

CURRENT IMMUNOASSAY METHODS

IMMUNOASSAY ISSUES RELATED TO PHYSIOLOGICAL VARIATION

IMMUNOASSAY ISSUES RELATED TO PRE-ANALYTIC VARIATION

IMMUNOASSAY ISSUES RELATED TO ANALYTIC VARIATION

tion (1,2) The majority of hormonal proteins and steroids are bioactive at

extremely low concentrations in the peripheral circulation, and hormonemetabolites often play a significant physiological role at target tissue sites There-fore, evaluation of a patient’s endocrine status with a given immunoassay isdependent upon both the sensitivity and specificity of that assay Hormone levelsoutside a given reference range can indicate a pathologic disease process Whenclinical decisions regarding a patient’s hormonal status or their response to atherapeutic intervention are based on hormone testing, it is important that dueconsideration be given to issues relating to the validity and utility of the assay

In practice, the performance of an immunoassay can be influenced by a variety

of physiological, pre-analytical and analytical factors, which may result in tion from the “true” value, i.e., that which reflects the actual physiological status

devia-of the patient Indeed, it is important to bear in mind that the clinical definition

of a “true” value for many hormones is relative only to the reference rangesestablished for the individual assay methods Furthermore, these issues oftenvitiate the ability to compare values generated by different laboratories

1

2 Gill et al.

The intent of this chapter is to provide the reader with an appreciation of how

an endocrine immunoassay can be an effective tool provided that the methods arecarefully chosen, monitored, and applied in the appropriate clinical setting

CURRENT IMMUNOASSAY METHODS

Principles

Immunoassays were originally developed as competitive binding assays thatutilized immunoglobulins as the binding protein The first clinically importantimmunoassay developed for hormones was the radioimmunoassay (RIA) RIA

is designed to utilize the competition, between hormone tagged with topes (the antigen) and unlabeled hormone in the patient specimen, for binding

radioiso-to a limited number of antibodies generated against that hormone (3,4) After

incubating the unlabeled specimens and standards (samples of known tion of a hormone) with assay reagents (antibody, radiolabeled hormone, buffer,and stabilizers), the amount of radiolabeled hormone bound to the antibody ismeasured The more unlabeled hormone present in the mixture, the less radiola-beled hormone will be bound to the antibody The standards provide a calibrationfor the amount of bound radiolabeled hormone across a range of known hormoneconcentrations, thereby allowing for interpretation of the assay results in terms

concentra-of clinically relevant hormone levels (5,6).

The accuracy of the RIA depends on the ability to separate antibody-boundfrom unbound reagents, particularly the radiolabeled antigen being measured Avariety of methods can be used to separate bound from unbound hormone afterthe competitive binding has occurred Historically, methods were used to pre-cipitate the bound antibody and separate the unbound reagents by centrifugation

(7) With the development of automated immunoassay systems, the antibody is

attached to solid materials, such as glass or latex beads, paramagnetic particles,

or plastic surfaces of tubes or small reagent cups (8–10) The antibody can be

immobilized to a solid material either directly, attaching to the surface during thebinding reaction, or indirectly, after the binding reaction has been completed.While the principle of the assay is the same in either case, there are significantdesign differences in these two approaches attributable to the kinetic relation-

ships between the reagents (11,12) Assays using indirect immobilization of the

antibody to a solid surface (for example, biotinylated antibody to avidin-coatedsurfaces) are analogous to RIAs, differing only in the method of separation Incontrast, in assays in which the antibody binds to the solid material during thebinding reaction, significant differences are observed in the kinetic properties ofthe antibody in solution vs attached to the solid material These assays aredesigned to present an excess amount of antibody in order to maximize hormonebinding both in solution and when immobilized to a solid surface Such systems

Chapter 1/Endocrine Immunoassay 3

are referred to as immunoradiometric assays (IRMA), rather than RIA, to size the differences between the design of these two approaches In both cases,the amount of hormone present in a patient specimen is determined by comparingthe amount of radioactive hormone bound to antibody in the specimen and in thestandards In addition to the standards and patient specimens, assays also includequality control specimens, which are standardized concentrations of hormonethat are tested in every assay to confirm its accuracy and precision

empha-While RIA is still considered a “gold standard” in reference laboratories, it hasnow been largely replaced by more rapid, nonisotopic methods, primarily enzyme-linked immunosorbent assays (ELISAs) There are many different design strat-

egies used for ELISAs (2,13,14) Early forms utilized competition between

hormone tagged with enzyme and unlabeled hormone This design is analogous

to RIA but substitutes enzyme activity for radioactivity for hormone detection.Enzyme activity is detected by washing away unbound reactants and then incu-bating the antibody–hormone complex with a substrate that produces a light-absorbing product More recently, substrates that produce chemiluminescence

upon enzymatic digestion have also been used effectively (15) Enzyme turnover

is then measured in a spectrophotometer or luminometer and is directly tional to the amount of enzyme-linked hormone that is bound As in RIA, theamount of labeled hormone is inversely proportional to the unlabeled hormone

propor-in the standards and unknown specimens

Immunometric immunoassays have now found widespread clinical utility,primarily for measurement of protein hormones These assays utilize multiple

antibody systems based on noncompetitive, e.g., “immunometric” designs (12).

In these systems, it is not necessary for the hormone to be chemically lated by labeling with radioactivity or enzyme Instead, endogenous hormone iscaptured with one antibody and quantitatively detected with another, which ischemically labeled with radioisotope (IRMA), enzyme, or luminescent chemi-cals, such as acrydinium esters The amount of detection antibody bound isdirectly proportional to the amount of antigen captured and can be used to gen-erate dose–response curves for assay standards As with all immunoassays, theconcentration of hormone in patient specimens is determined by comparingpatient results to the dose–response curves developed from the assay standards

manipu-Selected Survey of Current Methods

Commercially available systems with different methodologies in endocrineimmumodiagnostics are widely used in clinical laboratories today (Table 1) Thetrend over the past decade has been toward automated nonisotopic systems.Automated systems not only decrease the turn-around time for laboratory results,but also support larger testing volumes and test menus In addition, the overallcost of testing is reduced, as reportable results can be obtained efficiently with

4 Gill et al.

less technical expertise Reagent kits still, however, play a key role where highlyspecific tests for closely related proteins are required or when highly sensitiveimmunoassays are needed for analytical characterization of relatively smallnumbers of specimens in specialist chemistry laboratories

IMMUNOASSAY ISSUES RELATED

TO PHYSIOLOGICAL VARIATION

Overview

Perhaps the greatest challenge associated with use of immunoassays to assessendocrine status is the dynamic change in peripheral hormone concentrations.Hormone regulation involves an interplay of feed-forward and feedback interac-tions at different levels of the hypothalamic-pituitary-target organ axes Interpre-

Table 1 Overview of Selected Current Methods for Hormone Immunoassay

Design Instrument Manufacturer Operation Separation Detection

Method a Method b Reagent Kits

DSL Manual P, CT, CW R, CLinco Manual P, CW R, CSerotec Manual CW C

Automated Systems

Architect Abbott Continuous Access MP ELAxSYM Abbott Continuous Access PF EL

ACS180 Bayer Continuous Access MP LCentaur Bayer Continuous Access MP LImmuno 1 Bayer Continuous Access MP LAccess Beckman Continuous Access MP C

Immulite 2000 DPC Continuous Access CB ELAdvantage Nichols Continuous Access MP L ELViros Eci Ortho Continuous Access CW ELElecsys Roche Continuous Access MP L

a

Codes: CB, coated-bead; CT, coated-tube; CW, coated-well; MP, magnetic particle; P,

precipitation; PF, particle-filtration.

b

Codes: C, colorometric enzyme-linked immunoassay; EL, enzyme-chemiluminescent

immunoassay, L, chemiluminescent immunoassay; R, radioimmunoassay.

Chapter 1/Endocrine Immunoassay 5

tation of immunoassay measurements thus requires an appreciation of thedynamic nature of hormonal secretion and must be based upon appropriate ref-erence ranges In addition, immunoassays may vary based on differences in thenature of protein biosynthesis and hormone metabolism, dictating that onlymethod-specific comparisons be made

Biosynthesis of Protein Hormones

The biosynthesis, secretion, and clearance of hormones all contribute to thephysiologic variation in their serum concentration across time and betweennormal individuals Gonadotropin hormones provide a clear, albeit extreme,example Gonadotropins are dimeric glycoprotein hormones composed of an α-and β-subunit The gene for the α-subunit is expressed in both thyrotrope andgonadotrope cells and directs the synthesis of an α-subunit protein, which can besecreted free (free α-subunit) or complexed with one of three β-subunits.Gonadotropes express genes for the β-subunits of both luteinizing hormone (LH)and follicle-stimulating hormone (FSH), while thyrotropes express a gene for thesynthesis of the thyroid-stimulating hormone (TSH) β-subunit The bioactivity

of glycoprotein hormones is determined by the β-subunit Thus, a dimer posed of α-subunit complexed with a FSH β-subunit has FSH bioactivity, whilethe same α-subunit complexed with the TSH β-subunit has TSH bioactivity.The α-subunit gene is also expressed in the placenta, where it can be secreted

com-as either the free α-subunit or as the dimeric protein hormone human chorionicgonadotropin (hCG), in which it complexes with the hCG β-subunit The β-subunits of hCG and LH closely resemble each other sharing 97 of 121 aminoacids Indeed, hCG expresses many LH-like activities at target tissues

α- and β-subunit proteins are subject to posttranslational processing byglycosylation and proteolysis, both of which are well known to influence immu-noassay measurements Both subunits are glycosylated, resulting in a multitude

of isoforms of each hormone, which differ in both the number and type of sugarmoieties attached Proteolytic enzymatic activity, either prior to or after secre-tion into the peripheral blood, can result in additional isoforms of these hor-mones, which can be either nicked (broken but still complexed into a dimericprotein) or truncated (protein sequence missing)

These aspects of the biosynthesis and posttranslational processing of ropins are associated with differences in the specificity and accuracy of all cur-rent immunoassays The presence of a shared α-subunit among gonadotropins,TSH, and hCG results in significant cross-reactivity in immunoassays, whichdepend on the specificity of the antibody for the α-subunit Many of the earlyRIAs utilized polyclonal antibodies, which primarily recognized the α-subunit.Almost all current automated immunoanalyzers utilize at least one monoclonal

gonadot-or polyclonal antibody directed at the α-subunit, often resulting in inconsistent

or conflicting results Similarly, the high degree of structural homology between

says (17).

Inaccuracies between methods, attributable at least in part to specificity issues,

can be quite substantial (18) For example, results of FSH measurements of an identical specimen (Ligand–Special Series; August, 2000) distributed in aliquots

to different laboratories by the College of American Pathologists, revealed levelsvarying by almost 2-fold due to method-based differences in gonadotropinimmunoassays of proven clinical utility (Table 2)

Regulated Secretion of Protein and Steroid Hormones

Physiological variation in serum hormone concentrations can significantlyimpact immunoassay utility Use of hormonal measurements to assess apatient’s physiological status relies upon the reference ranges established foreach immunoassay For many hormones, including growth hormone (GH),adrenocorticotrophic hormone (ACTH), cortisol, and gonadotropins, estab-lishing reference ranges is confounded by their dynamic secretion, such thatthere is considerable variability from minute-to-minute, day-to-day, and overthe lifetime of an individual

Table 2 FSH Levels Measured in the Same Specimen by Different Laboratories

Using Various Commercial Immunoassay Systems

No.

Hormone Instrument Manufacturer Labs Low High Mean SD CV

FSH Architect Abbott 32 28.5 35.9 33.05 1.69 5.1%

AxSYM Abbott 561 26.9 38.9 32.63 2.09 6.4% Imx Abbott 28 30.4 35.8 32.82 1.57 4.8% ACS180 Bayer 219 38.2 52.5 45.81 2.66 5.8% Centaur Bayer 123 37.6 51.0 44.46 2.31 5.2% Immuno 1 Bayer 98 33.4 39.4 36.18 1.30 3.6% Access Beckman 102 32.0 43.1 38.02 2.19 5.8% Immulite DPC 106 33.4 44.4 38.51 2.23 5.8% Immulite 2000 DPC 37 31.3 41.3 36.39 2.39 6.6% IRMA (CAC) DPC 14 33.9 39.8 36.12 1.70 4.7% Elecsys Roche 49 32.8 47.0 38.91 3.60 9.3% Eci Vitros 45 23.8 28.7 25.69 1.22 4.7% DELFIA Wallac 5 26.5 31.2 — —

Chapter 1/Endocrine Immunoassay 7

P ULSATILITY

Neuroendocrine regulation of hormones involves a complex integrated work of feedback mechanisms between the hypothalamus, pituitary, and targetorgans In the reproductive axis, gonadotropin-releasing hormone (GnRH) issecreted in a pulsatile fashion by hypothalamic neurons, which in turn stimulatespulsatile secretion of LH and FSH by the gonadotrope cells of the anterior pitu-itary In young healthy men, pulses of LH are secreted approx every 120 min, and

net-given this pulsatile secretion, concentrations of LH can vary 3–4-fold (19) In

women, there are dynamic changes in the hypothalamic-pituitary-ovarian axis

across the menstrual cycle (20–22) LH pulses occur every 90 min in the early

follicular phase, increasing to every 60 min in the mid-follicular phase and then,following the mid-cycle LH surge and ovulation, slow progressively to 1 pulseevery 4 h in the late luteal phase Although secretion of most gonadal steroids isapulsatile, progesterone concentrations have been shown to fluctuate rapidly in

the luteal phase increasing from 2 to 40 ng/mL within minutes (22,23) Other

pituitary hormones, such as GH and TSH, are also secreted in a pulsatile manner,regulated by their respective hypothalamic releasing hormones, growth hor-mone-releasing hormone (GHRH) and thyrotropin-releasing hormone (TRH),respectively Such variability in serum hormone levels makes it difficult tointerpret a single blood test

D IURNAL AND C YCLIC V ARIATION IN H ORMONE L EVELS

The secretion of many hormones, including cortisol, TSH, GH, and prolactin,follows a circadian pattern The nocturnal rise of TSH begins in the early evening,peaks near midnight, and reaches nadir levels by early morning Conversely,cortisol levels peak in the morning and decline throughout the day, with lowestlevels achieved in the late afternoon–early evening GH and prolactin concentra-tions increase at night at the onset of sleep and decline by early morning Glucoseresponse to food also increases in the evening, which, along with a decline ininsulin secretion, results in decreased glucose tolerance in the late evening Anincrease in the counter-regulatory hormones at this time of day may contribute

to this relative insulin resistance

During puberty, there is a nocturnal rise in LH pulse frequency and amplitude

In adult men, mean gonadotropin levels remain relatively constant However,

LH pulse patterns may be quite variable in some individuals, resulting in a widerange of testosterone concentrations In one study, where blood was sampledevery 10 min for 24 h, 15% of young healthy men had serum testosterone levels

as low as 100 ng/dL documented following long interpulse intervals of LH

secretion (19) Testosterone also has a diurnal pattern with levels falling by approx 30% in the late afternoon and peaking in the early morning (19,24).

In women, there are dynamic changes in reproductive hormone levels across

the ovulatory menstrual cycle, which typically lasts approx 28 d (22,25–27) LH