Brown 2005

ENDOCRINE MANUAL FOR THE REPRODUCTIVE ASSESSMENT OF DOMESTIC AND NON-DOMESTIC SPECIES

Janine Brown, Ph.D. Susan Walker, M.S.

Karen Steinman, B.S. Conservation & Research Center National Zoological Park Front Royal, VA ©2005 CRC Endocrine Laboratory. All rights reserved.

Endocrine Manual – Brown et al. ©2005 ii

TABLE OF CONTENTS Page I. Introduction 1 II. Principles of Immunoassays 1 III. Principals of Enzymeimmunoassays 2 Essential components of an EIA 2 Steps to development of an EIA 3 IV. Types of Enzymeimmunoassays 4 Single antibody EIA 4 Double antibody EIA 5 Determining appropriate antibody dilution 7 Effect of antibody dilution on standard curve 7 V. General Assay Terminology 8 Antibody characteristics 8 Assay characteristics 9 Types of assay variation 9 VI. Monitoring Quality Control 10 Why is quality control necessary? 10 Proper use of the standard curve 10 Monitoring standard curve parameters 10 Monitoring assay quality 10 Example – Cortisol EIA control monitor 13 VII. Laboratory Immunoassay Validation 14 Parallelism 14 Recovery/Accuracy check 15 Extraction efficiency 17 VIII. Biological Validation of Methods 18 IX. HPLC for Analysis of Metabolites 19 X. Immunoassay Troubleshooting 20 Color and curve problems 20 Loss of binding 20 Shifted standard curve 21 High non-specific binding 21 Poor precision and high CVs 21 XI. Sample Collection/Storage Methods 23 Blood 23 Urine 24 Feces 24 Fecal dilutions 24 XII. Hormone Extraction Methods 25 Blood 25 Urine 25 Conjugated steroid analysis 25 Fecal extraction methods 26

Endocrine Manual – Brown et al. ©2005 iii

Extraction efficiency protocol 28 Fecal extraction sheet 30 XIII. Calculating EIA Results 31 Reading the plate 31 Calculating results from OD 31 Average blank value 31 Data matrix/table OD 32 Converting data from feces 35 Converting data from urine 36 Tips for reading EIA plates 36 XIV. Assay Protocols 37 Creatinine assay 37 Checkerboard titration for EIA 39 Cortisol EIA 42 Pregnane EIA 45 Testosterone EIA 48 Estrone conjugates EIA 51 XV. Assay Reagents and Supplies 54 Steroid EIA recipes 54 Creatinine assay recipes 55 Assay reagents for steroid and LH EIA 56 General supplies 57 General equipment 58 Other 59 XVI. Review of Steroid Metabolism 60 XVII. Review of Reproductive Physiology 69 XVIII. Review of Adrenal Metabolism 71

Endocrine Manual – Brown et al. ©2005 1

I. INTRODUCTION Immunological techniques, like radioimmunoassays (RIA) and enzyme immunoassays (EIA), are used because they are capable of measuring small quantities of hormones. RIAs are highly sensitive and have been the most common immunological methods for hormone analysis used to date. However, an RIA laboratory needs to be licensed for the use of radioisotopic tracers, and gamma and beta scintillation detection equipment are comparatively expensive. By contrast, EIAs do not utilize radioactivity, equipment is less expensive and reagents are easy to prepare, are highly stable and have a long shelf-life. Many EIAs are now as sensitive as RIAs and so are gaining in popularity. The purpose of this manual is to acquaint the reader with both RIA and EIA techniques; however, the emphasis will be on EIA because of its universal adaptability and potential for development as field tests. II. PRINCIPLES OF IMMUNOASSAYS The essence of an immunoassay is the competition between added labeled antigen (‘tracer’) and unlabeled antigen (i.e., hormone in the sample) binding to an antibody. Highly sensitive immunoassays rely on the use of a limited amount of antibody. If the primary antibody is in excess, there is little or no competition in binding between labeled and unlabeled antigen, thus no discrimination in measuring concentration of the unknown. With a limited amount of antibody, samples with higher concentrations of hormone have a greater chance of competing against labeled antigen for antibody binding than low concentration samples, and this relationship is proportional to the amount of unlabeled hormone added. Antigen-antibody binding in RIA and EIA follows the Law of Mass Action. The distribution between the bound and unbound phases is directly related to the total amount of antigen (Ag) in the presence of a fixed amount of antibody (Ab), where k1 and k2 denote the association (forwards reaction) and dissociation (reverse reaction) constants, respectively. k2 Ag + Ab � AgAb k1 In the beginning, the rate of the reaction is greater in the forward direction (k1) until equilibrium is achieved; at equilibrium there will be no further net change in the concentrations on either side of the equation. The affinity constant: K = k1/k2 High K implies that the reaction is favored in the forward direction; low K implies the reaction is favored in the opposite direction. Given a constant amount of antibody of fixed K, the ratio of bound to free antigen will be related to the total amount of antigen present. Antibodies are produced against a specific antigen by immunizing an animal and collecting immune serum.

• Primary antibody (or antisera), also called the “first antibody”, refers to the hormone-specific antibody; the one that was produced from immunizing an animal against the hormone to be measured. Primary antisera can be produced against large proteins by direct injection of antigen solubilized in a carrier that stimulates the immune response (i.e., Freund’s adjuvant). Immunization against small proteins or steroids (i.e., haptens) requires conjugation to immune stimulating molecules (i.e., albumins, keyhole limpet antigen, etc.). Primary antibodies are often produced in rabbits, guinea pigs and monkeys.

• Second antibodies are those produced by immunizing a different species like a goat or sheep against IgGs of the species that produced the primary antisera (e.g., goat anti-rabbit antisera). The second antibody is not specific for the primary antigen, but is used in sandwich assays.

• Polyclonal antibodies are produced by injecting an animal with a purified or partially purified antigen and collecting the immune serum. The antisera produced contains a variety of immunoglobulins against the antigen (or antigens for partially purified immunogen) or conjugate molecule (in the case of conjugated haptens). Antibody production is limited to the life span of the immunized animal and success of re-immunizations.

• Monoclonal antibodies are produced by immunizing an animal (e.g., mouse) and cloning individual antibody cells to produce a single variant antibody against an antigenic determinant on the hormone. Antibody production is indefinite as long as the cloned cells are maintained (frozen in liquid nitrogen).


III. PRINCIPLES OF ENZYMEIMMUNOASSAY EIA is also known as ELISA (Enzyme Linked ImmunoSorbent Assay). EIAs depend on the assumption that an antigen can be linked to an enzyme and retain both immunological and enzymatic activity in the resultant conjugate. The soluble antigen or antibody must also be linked to an insoluble phase in a way in which the reactivity of the immunological component is retained. ESSENTIAL COMPONENTS OF THE EIA:

• Solid Phase: The solid phase is the polystyrene microtiter plate.

• Antibody: An immunoglobulin produced against a specific antigen. Polyclonal antibodies must be affinity purified for EIA.

• Coating buffer: The antibody is diluted with an alkaline buffer, usually a carbonate/bicarbonate buffer of pH 9.6, which causes it to passively adsorb to the well of the microtiter plate.

• Wash solution: Each incubation is terminated by a washing step. The wash removes all unbound components from the plate.

• Enzyme conjugate (tracer): The enzyme conjugate is the component of the assay that permits detection of antigen concentration. For direct, single antibody EIAs, a common enzyme conjugate is hormone conjugated to horseradish peroxidase (HRP). For double antibody sandwich EIAs, the enzyme conjugate complex is a biotin labeled hormone that binds to peroxidase-labeled streptavidin.

• Assay buffer: Phosphate or Tris buffers of pH 7.0 are commonly used. Sodium azide cannot be used in buffers for single antibody EIAs because the HRP is inhibited by azide. Sample dilutions, standards and enzyme conjugate are made up in assay buffer.

• Standards or unlabeled antigen: The standard is usually the same antigen that was used to make the antibody and the same as the enzyme conjugate, or is structurally similar so that it crossreacts with the first antibody. Standards are used in a series of known concentrations against which unknown concentrations of antigen in the sample can be measured and calculated.

• Substrate: The substrate reacts with the bound enzyme conjugate and changes color. It consists of three components: buffer, chromagen, and catalyst. The buffer has an acidic pH and is either citric acid or phosphate citrate buffer. The chromagen is the color changer and is usually azino-bis-3-ethyl benzthiazoline-6-sulfonic acid (ABTS) or tetramethylbenzadine (TMB). ABTS turns a green color and TMB turns blue. The catalyst is what causes the reaction, via oxidation-reduction, and is hydrogen peroxide or sodium perborate.

• Stop solution: Sulfuric acid solution that stops the substrate reaction and allows the plate to be read at any time. It is used primarily in the double antibody EIA. It causes the blue substrate to turn yellow.

STEPS TO DEVELOPMENT OF AN EIA

1. Determination of antibody titer. An appropriate antibody titer is one that results in adequate color change while retaining good sensitivity. In general, increasing the antibody concentration increases the enzymatic color change, but decreases assay sensitivity. Decreasing antibody concentration (more dilute) increases sensitivity, but the color change is less.

2. Determination of enzyme conjugate dilution. Increased enzyme conjugate concentration results in a stronger color change but decreased assay sensitivity, whereas decreased conjugate concentration increases assay sensitivity but reduces color intensity. An appropriate combination of antibody and enzyme conjugate results in adequate color intensity with high assay sensitivity.

3. Development of standard curve. Incubation of a fixed amount of enzyme conjugate and antibody in the presence of different concentrations of standard (unlabeled antigen). A graph is generated that depicts the relationship between the percentage of bound enzyme conjugate (relative to the maximum binding of the enzyme conjugate, zero well) to the concentration/mass of the standard added. The relationship of the percent binding and the standard mass is inversely proportional.

4. Time and temperature of incubation. Incubation time can be decreased with increased temperature but antibody-antigen binding and substrate-enzyme conjugate binding can decrease if the temperature is too high.

5. Definitions:


• Total Binding (TB): Maximum binding of enzyme conjugate (labeled antigen) to the antibody in the absence of competition from unlabeled antigen (standard or unknown sample). Also called “zeroes” and should have the highest optical densities. It is assumed to be 100% binding of the enzyme conjugate. Most assay systems attempt to reach optical densities of 0.8-1.0 in the zero wells.

• Unknowns: Unknown hormone concentrations in samples are determined by comparing the specific binding of the samples to the binding obtained from standard hormone concentrations of known mass.

• Non-specific binding (NSB): Amount of binding that occurs in the plate that is NOT due to the antibody, but to other components of the assay. It also accounts for the amount of interference the plastic bottom of the plate produces when the light from the plate reader is refracted. The NSB wells should be less than 10% of the maximum binding wells (zeroes). The NSB results are subtracted from the sample/standard results (this is done by the microplate reader). NSB can be reduced by adding a protein blocking buffer after antibody coating, adding detergent to the assay buffer or increasing the number of washes.


IV. TYPES OF ENZYMEIMMUNOASSAYS SINGLE ANTIBODY EIA (e.g., cortisol) A hormone-specific antibody (or first antibody) is passively adsorbed (i.e., coated) to a polystyrene microtiter plate. Unabsorbed antibody is washed away. Known (standards) and unknown (samples) concentrations of hormone (unlabeled antigen) and the hormone-specific enzyme conjugate (HRP) (the labeled antigen) are added to the well. The labeled and unlabeled antigens compete for binding sites on the antibody during the incubation phase. The unbound components are washed away. The substrate is added and reacts with the bound enzyme conjugate and changes color. The more color change in the well, the more enzyme conjugate is bound, meaning less hormone. The relationship of color to hormone concentration is inversely proportional. The zero wells contain only enzyme conjugate so there is no competition for antibody binding. The zero wells represent the maximum binding of the labeled antigen and, hence, have the most color change. Example: Y First antibody HRP-labeled hormone Free hormone Substrate

1) Antibody binding to solid phase (known as coating)

Y Y Y Y Y Y Y Y

2) Competition for antibody binding sites by labeled and sample hormone

Y Y Y Y Y Y Y Y

3) Wash away excess unbound hormone

Y Y Y Y Y Y Y Y

4) Substrate binding to HRP-labeled hormone

Y Y Y Y Y Y Y Y

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Zero standard High standard Maximum color Minimum color


DOUBLE ANTIBODY EIA (e.g., luteinizing hormone) In this antibody system a ‘second antibody’ (i.e., anti-first antibody) that recognizes the first antibody and other IgGs is used to coat the microtiter plate. Following incubation, unbound second antibody is decanted and a blocking buffer, usually containing a protein such as BSA is added to reduce non-specific binding. After incubation, plates are washed and any unbound components removed. The first antibody and unlabeled antigens (sample or standards) are added to the wells. The first antibody binds to the second antibody and the free hormone then binds to the first antibody. Reagents are allowed to incubate followed by incubation with the biotin-labeled antigen. Plates are washed, removing any unbound biotin-labeled antigen, and streptavidin-peroxidase is added. The distinguishing feature of the avidin-biotin system is the extremely high affinity of the avidin (from egg white or a bacteria -Streptomyces avidinii) for biotin (a water-soluble B vitamin). The speed and the strength of binding between these two molecules is used to provide an amplification of the enzyme signal. This binding also is not influenced as much by extreme temperatures or pH levels. The peroxidase enzyme is incorporated into the streptavidin and after binding to the biotin, forms the enzyme conjugate complex. Following plate washing substrate (TMB) is added. The chromagen within the substrate reacts with the bound enzyme conjugate and changes color. Similar to the direct single antibody competitive EIA, the more color change in the well the more enzyme conjugate bound, meaning less hormone. The relationship of color to hormone concentration is inversely proportional. Example: Second antibody (recognizes IgGs) BSA Y First antibody (recognizes hormone antigen) Biotin-labeled hormone

Substrate Free hormone Streptavidin-peroxidase (enzyme)

1) Second antibody (anti-first antibody) binding to solid phase (coating) 2) BSA is added to block non-specific binding sites


3) Addition of hormone-specific, first antibody, biotin-labeled hormone, and free hormone

Y Y Y Y

4) Wash away unbound components

Y Y Y Y 5) Addition of streptavidin-peroxidase (enzyme)

Y Y Y Y 6) Addition of substrate

Y Y Y Y 7) Results Y Y Y Y Y Y Y Y Zero High standard Maximum color Minimum color


HOW TO DETERMINE APPROPRIATE ANTIBODY DILUTION FOR EIA

ŹŹŹŹŹŹ

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Ab Dilution (x 1000)ŹŹŹ

% Binding

(TB/TC)Want Ab to bind ~30%

of labeled hormone

Use Ab at 1:120,000 dilution

Appropriate first antibody concentration is determined by running a titration curve, which involves incubating serial dilutions of first antibody with a constant amount of HRP. Calculate the % binding of the antibody to tracer and plot as a linear-log curve. The best antibody dilution (one that provides good sensitivity with adequate detectibility) is ~30% (range 20-50%). EFFECT OF ANTIBODY DILUTION ON STANDARD CURVE CHARACTERISTICS

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Standard ConcentrationŹŹŹ

% B/TB

Correct

too much

not enough

Inappropriate first antibody concentration will result in a poor standard curve that is too ‘flat’ on the top or

bottom portion. Too much antibody also will reduce assay sensitivity.


VII. GENERAL ASSAY TERMINOLOGY ANTIBODY CHARACTERISTICS Sensitivity: The ability to detect small quantities of antigen. It is the lowest concentration of antigen

that can be statistically distinguished from a sample with no antigen. - objective determination - calculated as the value 2 SD from the mean response of

the blank or zero (Bo) tube. - subjective determination - the value at 90% or 95% of maximum binding.

Specificity: The ability of the antibody to discriminate between antigens. Crossreactivity: The ability of an antibody to have immunoreactivity with a more than one antigen.

Crossreactivity of an antibody with other hormones is determined using binding inhibition curves. It is expressed as the standard concentration at 50% binding divided by the concentration of the competitive antigen at 50% binding, expressed as a percentage.

Examples:

• Hormone C is detected but the antibody is not as specific for it. The crossreactivity is less than 100%.

• The antibody for progesterone crossreacts better with hormone D than the standard. The crossreactivity is over 100%.

• Hormones A and B only bind the antibody at high concentrations and crossreactivity cannot be calculated.

Crossreactivity of Different Antigens on a Progesterone EIA

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Standard Concentration (ng)

% B/TB

Progesterone Standard

Hormone A

Hormone B

Hormone C

Hormone D


ASSAY CHARACTERISTICS Accuracy: The degree to which the measured concentration corresponds to the true concentration of a

substance. This is related in part to the specificity of the assay. Precision: Refers to the repeatability of a measured value or the consistency of results. It is a measure of

random error defined as the variation among replicate measurements of a defined sample. It is expressed as the Coefficient of Variation (%CV) which is the (Standard Deviation/Mean)*100.

TYPES OF ASSAY VARIATION

Intra-assay variation: The variation within assays determined by repeated analysis of samples, typically low,

medium and high, within a single assay. Inter-assay variation: The variation between assays determined by repeated analysis of the same sample in

several assays. These samples are called controls and should bind at 30% and 70%.

Note: A sample can be precise, but not accurate. This occurs when the measured value deviates from the true value as a result of using a non-specific first antibody or to systematic error, like improperly calibrated weighing or measuring equipment, improper technique (pipetting) or sample problems.


VIII. MONITORING QUALITY CONTROL WHY IS QUALITY CONTROL NECESSARY?

• Proper validation and standardization of an assay is only the first step towards establishing a reliable endocrine monitoring program. Subsequent assessment of assay quality and consistency is absolutely necessary to assure the biological relevance of results.

• For every assay system there is an inherent level of error which must be accepted.

• A quality control program indicates when that level of error becomes unacceptable. • Quality control samples only have value if their analysis provides a reasonable confidence in data for

the whole assay, or reflects a true error in the method.

• It is an ongoing process; the value of quality control values increases with time. PROPER USE OF THE STANDARD CURVE

• Assay range (the 20-80% rule): A subjective method of sample exclusion which eliminates the use of values in which the binding is less than 20% or greater than 80% of maximum binding. All outliers are re-analyzed after appropriate modification (i.e., taking more or less sample to the assay). This rule is based on the assumption that most standard curves are linear between 20 and 80% binding, and therefore the dose response is linear. The binding cut-off limits may vary, however, and should be appropriate for each assay.

MONITORING STANDARD CURVE PARAMETERS

• Total binding (%B/Bo): Indicates the maximum binding of label in the given assay system. Given that the assay parameters are unchanged, the maximum binding should remain relatively constant from assay to assay. A decrease in binding suggests one or more assay factors are not optimized (see Troubleshooting).

• Non-specific binding (%NB/Bo): The amount of binding due to factors other than specific antibody binding. It should remain constant from assay to assay. It also should be kept to a minimum, generally less than 5% of the total counts added. An increase in NSB again suggests one or more assay factors are not optimized (see Troubleshooting).

• Effective dose values (ED20, ED50, ED80): Gives a good estimate of the overall ‘shape’ of the curve. The ED50 is especially useful for indicating changes in assay sensitivity. The ED20 and ED80 can also indicate changes in the slope of the curve.

MONITORING ASSAY QUALITY

• How is quality control monitored? Assay consistency is monitored by analyzing ‘internal control samples,’ which are treated as unknowns, but are run in every assay.

- how many to run? Usually 2 - 3 controls, assayed in duplicate or triplicate. Much of this depends upon the variability of the assay.

- what concentrations to run? Controls should provide an estimate of variability over the working range of the standard curve. Recommend controls be run at ~30% (high concentration), 50% (medium) and 70% (low) of maximum binding.


- what samples can be used as controls? Almost any biological material containing the relevant antigen, including serum pools, fecal extracts, purified steroids diluted in buffer or other fluid (provided it does not interfere with the assay), pituitary extracts, urine pools, purchased standards, etc.

1. prepare a large pool projected to last for several years. 2. divide the material into small aliquots to avoid repeated freeze-thawing. 3. keep the aliquots in a safe freezer (preferably one with a temperature alarm). 4. store all materials, including samples, in a freezer that is NOT frost- free. 5. do not allow controls to run out before making up new controls.

• Assay coefficients of variation: (%CV = standard deviation/mean times 100) - intra-assay CV - determines the within assay error (the error associated with running the same

sample in one assay). Most accurately determined by calculating the variation in assaying multiple replicates of one sample throughout the assay (e.g., n = 10 replicates). More typically, the average intra-assay CV is calculated from the internal controls assayed at the beginning of the assay. A third method is to calculate the average CV of all unknowns run in an assay. If more than one assay has been run for a particular study, then the mean intra-assay CVs for those assays should be averaged.

- inter-assay CV - determines the between assay error (the error observed when the same sample is run in different assays). Determined by calculating the variation in values for samples run in every assay. Within a study can calculate the individual inter-assay CVs for each internal control and then average those numbers.

• Causes of variation: - intra-assay CV - generally is the result of the presence of unequal amounts of sample, tracer or

antibody (i.e., poor pipetting, or incomplete mixing of sample or reagents), but also can be due to inconsistencies in counting or decanting.

- inter-assay CV - caused by system-related problems like reagent instability, procedural variation or changes in standards (do not allow old standards to run out before making up new standards!).

• What is an acceptable level of error? - sample rejection - a level of error needs to be established that objectively determines when a

sample needs to be re-analyzed. However, the amount of error tolerated is subjective and determined, in part, by how critically the data need to be interpreted. For example, the error rate would be lower in a human endocrinology laboratory where health status and potential treatments hinge on accurate measurements. Conversely, more error might be tolerated in assays conducted to define hormonal trends (i.e., the absolute values are not as important as the profile).

- rules of thumb for defining acceptable levels of error 1. fixed percentage of the CV - often designated as 10% such that all samples with a CV

>10% are re-analyzed. 2. assay rejection criteria - often subjective, but can involve exclusions based on control

values falling within 2 SD of the mean of previous values, or a set proportion of sample CVs being below 10%.


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Standard Concentration

% B/TB

The same 5% error in binding results in a greater %CV at each

end of the curve

• Proportional error: The CVs are higher and dose calculations are not proportional at the extreme ends

of the standard curve, due to the non-linearity of the curve (see above figure). • External quality control procedures: Commonly used in clinical laboratories to ensure that the quality

of data are independent of the laboratory, which generated it. Involves analysis of samples provided by external sources.


Cortisol EIA Control Monitor Controls Standards

Date Tech QCH %B

QCH Dose

QCL %B

QCL Dose 7

.8

15.6

31.3

62.5

125.0

250.0

500.0

4/22/02 Sw 74.8 64.4 53.7 114.9 100.0 101.2 89.6 74.7 51.9 23.6 9.2 6/27/02 Sw 69.8 57.4 48.0 106.6 92.6 88.7 82.2 67.5 42.2 16.9 6.4 6/28/02 Sw 66.4 58.6 46.6 104.0 98.5 90.4 80.6 65.1 42.0 14.8 5.9 6/29/02 Sw 70.5 57.1 47.8 106.6 99.0 92.2 83.9 67.1 42.7 16.1 6.2 6/30/02 Sw 68.3 58.6 44.8 114.0 91.6 81.0 67.7 42.5 15.7 5.7 7/1/02 Sw 63.0 68.5 44.5 114.1 99.0 90.9 80.7 66.0 42.2 15.7 5.2 7/1/02 Sw 68.4 59.8 49.1 102.5 101.0 94.0 83.0 67.2 43.0 17.0 6.7 7/1/02 Sw 73.4 44.8 50.7 94.3 91.5 88.7 79.8 64.7 42.0 16.3 6.0 7/1/02 Sw 67.3 61.3 48.2 104.0 99.2 91.1 82.0 66.4 42.4 15.8 6.0 7/3/01 Sw 71.6 50.0 47.0 102.8 96.7 92.6 83.4 63.4 41.6 16.7 6.3 7/3/01 Sw 69.7 52.1 45.7 102.3 98.0 91.0 81.8 64.6 38.7 15.2 5.1 7/3/01 Sw 71.9 54.4 46.3 108.7 93.9 94.7 83.1 66.8 41.4 16.4 6.1 7/3/01 Sw 68.8 62.7 48.7 107.7 104.1 94.2 87.1 69.9 43.9 16.7 4.8 7/19/02 Sw 64.8 53.4 42.0 104.6 96.5 87.4 77.7 60.8 36.3 13.3 3.1 7/19/02 Sw 68.1 53.3 46.3 106.5 96.6 88.8 79.7 64.0 44.9 12.7 3.8 7/19/02 Sw 69.6 47.4 43.0 102.3 101.1 91.5 78.6 64.0 35.4 13.1 4.0 7/19/02 Sw 66.7 50.7 40.3 112.0 97.2 85.3 78.2 62.8 36.8 13.1 4.3 7/31/02 Sw 68.6 59.6 49.8 105.8 96.3 88.1 81.0 67.9 44.8 20.6 12.8 7/31/02 Sw 72.3 50.1 48.4 104.4 97.7 88.2 81.7 68.1 42.6 18.8 11.3 10/23/02 Sw 73.4 54.0 51.4 103.2 102.0 91.7 85.2 70.4 45.3 17.7 5.6 10/23/02 Sw 75.7 50.8 55.1 96.9 103.0 91.4 86.2 69.8 48.0 17.4 4.0 Mean 69.7 55.7 47.5 105.6 98.2 91.1 82.2 66.6 42.4 16.4 6.1 Count 21.0 21.0 21.0 21.0 20.0 21.0 21.0 21.0 21.0 21.0 21.0 Std Dev 3.2 5.9 3.6 5.2 3.2 3.3 3.0 3.1 3.7 2.5 2.4 CV 4.6 10.6 7.6 4.9 3.3 3.6 3.6 4.6 8.8 15.5 38.7

Mn +1SD 72.9 61.6 51.1 110.8 101.4 94.4 85.2 69.7 46.1 18.9 8.5

Mn-1SD

66.4 49.7 43.9 100.4 94.9 87.8 79.2 63.5 38.7 13.8 3.7

Min 63.0 44.8 40.3 94.3 91.5 85.3 77.7 60.8 35.4 12.7 3.1 Max 75.7 68.5 55.1 114.9 104.1 101.2 89.6 74.7 51.9 23.6 12.8


IX. LABORATORY IMMUNOASSAY VALIDATION PARALLELISM Definition: Parallelism is a way of determining if the assay is actually measuring what it should be measuring. It can also tell what dilution of sample to use for the assay.

• Samples to test should reflect the range of normal concentrations expected (i.e., estrous cycle and pregnancy, seasonal samples).

• Pool an equal amount of urine or fecal extract from each sample and dilute serially two-fold in assay buffer (neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, etc.). For example add 200 µl of buffer into tubes labeled from 1:2 to 1:8192 - then take 200 µl of the neat sample and mix with the 200 µl in the “1:2” tube, vortex the “1:2” - then take 200 µl out of the 1:2 and place in the “1:4” and so on. This will result in 200 µl in each tube except the last dilution, which will have 400 µl.

• Plot the % binding of samples by choosing an arbitrary concentration for the neat sample and halving the concentration for each dilution.

• If the sample curve parallels the standard curve the sample hormone is immunologically similar to the standard and can be measured proportionately.

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Standard Concentration or DilutionŹŹŹŹ

% B/TB

Standard curve

Parallel displacement

Use 1:16

neat

1:4

1:641:32

1:8

1:2

The best (most accurate) dilution to run samples is at ~50% binding (see above figure). The pool shows parallelism with the standard curve and is therefore valid for use in the assay. Samples should be run at a 1:16 dilution based on the binding inhibition observed at ~50%.


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Standard Concentration or DilutionŹŹŹŹ

% B/TB

Standard curve

No displacement

Non-parallel displacement

Low concentration

In the graph above, samples that produced non-parallel or no displacement cannot be run in this assay system because they do not demonstrate immunoactivity of endogenous antigen similar to the assay standards. The sample with a low concentration of immunoactive antigen demonstrated limited parallelism, but this type of sample could only be run ‘neat’ and there likely would be problems with limited detection within the working range of the assay (i.e., <80% binding). RECOVERY/ACCURACY CHECK Definition: A recovery tests for potential interference caused by substances contained within the biological sample that are independent of specific antigen-antibody binding. This test indicates the degree to which the measured concentration corresponds to the true concentration of a substance.

• Make a sample pool. It should have a low concentration of hormone (e.g., for progestagens use samples from the follicular phase).

• Spike aliquots of pooled sample (i.e., 100 µl) with an equal amount (i.e., 100 µl) from each standard.

• Analyze the spiked samples as unknowns in the assay.

• The sample pool also needs to be analyzed without added standard to determine the amount of endogenous (or background) hormone present. That will have to be subtracted out from each spiked sample.

Results: Amount Expected = (Concentration of standard spiked with / 2) Amount Observed = (Concentration observed from assay results – minus background) % Recovery = (Amount Observed/Amount Expected)*100


Example:

Standard concentration (pg/well)

Amount added - Amount expected (pg/well)

Amount observed (pg/well)

Minus background (pg/well) % Recovery

500.00 250.00 235.48 218.64 87.46

250.00 125.00 119.07 102.23 81.78

125.00 62.50 65.85 49.01 78.42

62.50 31.25 42.52 25.68 82.18

31.25 15.63 30.10 13.26 84.86

15.62 7.81 23.45 6.61 84.64

7.80 3.90 19.98 3.14 80.51

3.90 1.95 18.54 1.70 87.18 Endogenous/ Background

0.00 16.84 Plot Amount Expected vs. Amount Observed - conduct a linear regression (y=mx+b) analysis. Slopes > or < 1 suggests an over or under estimation of hormone mass, respectively.

Elephant Urine PdG RecoveryŹŹŹŹŹŹ

y = 0.8679x + 15.343

R2 = 0.9986

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Amount ExpectedŹŹŹ

Amount Observed


EXTRACTION EFFICIENCY Definition: A way of defining how efficient or effective the extraction method is at ‘extracting’ or pulling out metabolites of interest (steroid or protein hormones) from a specific matrix (urine, fecal, blood or saliva). Monitoring extraction efficiency also permits monitoring how consistent the extraction process is from sample to sample.

• Similar to recovery but samples are spiked before extraction.

• Spike each sample with:

Labeled hormone: ~2000 dpm/100 µl of 3H or

14C radiolabeled steroid

or Unlabeled (cold) hormone: (mass is dependant upon sensitivity of assay system) – add enough mass to read at about 50% binding

• Extract sample as per protocol. After extraction:

• Determine “Amount Observed”: Labeled hormone: aliquot 50 µl from the final 1.0 ml extract to scintillation vial and count radioactivity.

or Unlabeled hormone: analyze by RIA or EIA to determine concentration.

• Determine “Amount Expected”: Labeled hormone: aliquot 100 µl of original stock of radiolabeled steroid to scintillation vial and count radioactivity.

or Unlabeled hormone: dilute standard stock the sample the sample was spiked with to determine concentration.

Results: Amount Expected = (expected minus background) Amount Observed = (observed minus background) % Extraction efficiency = (Amount Observed/Amount Expected)*100


X. BIOLOGICAL VALIDATION OF METHODS

It is crucial to demonstrate that hormonal measures accurately reflect the physiological events of interest. From a practical standpoint, it is unnecessary to determine the specific molecular structure of the hormones being monitored in each species. However, it is critical to prove that fluctuations in the hormonal metabolite being measured provide physiologically relevant information.

Validation based on physiological events

As examples, an ovarian cycle might be validated by comparing two independent measures of the same hormone in matched samples (i.e., fecal vs. urinary estrogen) or by comparing temporal hormonal excretion patterns with external signs of reproductive status (e.g., estrogens should increase with sex skin swelling, estrus, copulatory behavior). Similarly, showing a predicted rise and fall in progestin metabolite coincident with pregnancy onset and parturition, respectively, is solid evidence of the assay’s utility for assessing pregnancy status. Validation using hormonal challenges Administering a drug known to stimulate hormonal production is also useful for demonstrating a cause-and-effect relationship between its exogenous administration and the subsequent excretion of the target hormone. Typically, hormone challenges include gonadotropin releasing hormone (GnRH) to study pituitary hormones (LH, FSH) and subsequent androgen production (in males), or adrenocorticotropic hormone (ACTH) and cortisol secretion. This approach also clarifies the excretory lag-time between stimulation of an endocrine gland and the appearance of its hormonal metabolites in excreta. General protocol for ACTH or GnRH challenges:

• GnRH (e.g., Cystorelin) is typically administered at a dose of 1 –5 �g/kg body weight; ACTH (gel form preferred) is given at doses of 2-5 IU/kg. Drugs can be administered i.v. (preferable), i.m. or s.c.

• If blood collection is possible, samples should be collected as follows: -20 min, -10 min and 0 min, with 0 = time of hormone injection; 15 min, 30 min, 45 min, 60 min, 90 min, 2 hr, 3 hr, 4 hr, 6 hr and 8 hr post-injection. It is important that at least two pre-injection samples be collected, and that several are collected for at least 6 hr after injection.

• For fecal hormone validation, several pre-injection samples should be collected and every fecal sample for at least five days post-injection.

• For urinary hormone validation, several pre-injection samples should be collected (can start collecting the week before the test) and every urine sample for at least two days post-injection.


XI. HPLC FOR ANALYSIS OF METABOLITES High performance (or pressure) liquid chromatography is a separation technique used to identify specific metabolites or components within a given sample. HPLC separates a sample into fractions, each of which is analyzed for crossreactivity in a given assay. The separation is performed when two phases are brought in contact, one phase being stationary (the column) and the other being mobile (a liquid). The sample mixture is forced through the column under pressure and undergoes a series of interactions between the stationary and mobile phases. Interactions exploit differences in the physical or chemical properties of the components within the sample. These differences govern the rate of migration of the individual components under the influence of the mobile phase moving through a column. Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Selection of the appropriate HPLC method is governed by sample properties such as molecular weight, solubility and polarity. The most common method for the separation of steroids is reverse phase chromatography and is based on polarity [a non-polar stationary phase (a column packed with hydrocarbons) and a polar mobile phase (eg., methanol, water, acetonitrile and ethanol)]. The sample components are attracted to the surface of the adsorbent (the solid phase) with differing strength. Non-polar samples are attracted to the stationary phase and are retained, the more polar compounds elute first. As the gradient of the mobile phase is adjusted over time components within the sample are separated. As the polarity of the mobile phase increases the non-polar samples begin to elude from the column.

HPLC of Fecal Estrone Metabolites

0

2000

4000

6000

8000

1 11 21 31 41 51 61 71

HPLC fraction

Radioactivity (cpm/fraction)

0

2000

4000

6000

8000

10000

Immuno-activity

(pg/fraction)

Known Antigens

Immunoreactivity on EIA

Estrone-3-Sulfate (Fraction 16)

Estrone (Fraction 63)

Unknown metabolite


XII. IMMUNOASSAY TROUBLE SHOOTING Some of the problems listed below are for EIA only. Those relevant to both EIA and RIA are designated by an asterisk “*”. COLOR AND CURVE PROBLEMS Color develops too quickly (or covers entire plate)

• Conjugate is too concentrated – use higher dilution

• Curve will be flat on top • This could also be the effect of too much first antibody

Color develops too slowly (or not at all)

• Conjugate is too weak – retitrate to lower dilution (add more) • Curve will be flat on bottom • This could also be the effect of too little first antibody

• Also, hydrogen peroxide could be bad (re-make fresh), diluted wrong or not added. • Also see Loss of Binding Section

Patchy color development

• See Poor Precision and High CV’s section LOSS OF BINDING Decomposition of the antibody*

• Some antibodies are more stable than others. Best to store antibody frozen at a set dilution in a no frost-free freezer, or preferably an ultra-low freezer (especially for long-term storage of stocks). Store antibody upright (to decrease surface area exposed) in o-ring vials to ensure a tight seal.

• Antibody solutions should be prepared carefully (e.g., do not expose to excessive heat or shake vigorously).

• Most problems with loss of binding are NOT due to the antibody unless it is because not enough antibody was added.

Poor label (or conjugate) characteristics*

• This is the most common factor causing low binding.

• Making fresh label will often correct low binding and improve color development. • Care must be taken with these reagents; they provide the signal for the assay.

i. Always store label at recommended temperatures. ii. Never store working label dilution for long periods of time. iii. It should always be made up just before use. iv. Never leave label on the bench at RT for excessive amounts of time.

Ineffective separation

• Wash plates the recommended number of times and maintain consistency across the plate. Improper washing will lead to high CV’s and loss of binding.


Reduced quality of assay reagents*

• It is important that buffers are made properly and often. Check buffer pH. Be sure that proteins like BSA are stored properly and not too old.

Calculation error*

• Take care that all reagent calculations are correct. This is an easy place to make a simple mistake! Keep records of all calculations for each assay.

SHIFTED STANDARD CURVE Parallel shift*

• Shift to the left - too much standard

• Shift to the right - too little standard Causes:

- Inaccurate weighing of standards - check balance calibration - Inaccurate diluting of standards - check pipette calibration - Contamination of standard stock - make new standard stock - Changes in standard stock - due to evaporation, adsorption to storage vessel, etc.

Non-parallel shift*

• Contaminated buffers - contamination with cross-reacting analytes or other compounds that interfere with binding.

HIGH NON-SPECIFIC BINDING*

Decomposition of label*

• Non-specific binding often increases as labels age or become damaged. Adherence of label to well or tube*

• Can be due to label decomposition or problems with buffer proteins that no longer prevent sticking. Incomplete removal of label*

• This can be caused by poor washing of the plate or decanting of assay tubes. Non-specific attachment of antibody

• Unsuitable blocking buffer used (needs to be re-made) or omission of blocking buffer (forgot to add it).

POOR PRECISION AND HIGH CVs* Human error*

• Inconsistent pipetting, washing etc. The key is to be consistent.

• Avoid bubbles in pipet tips.

Incomplete mixing of samples or other reagents

• All frozen samples must be thoroughly, but gently, mixed after thawing. Never shake samples or assay reagents vigorously.

Equipment failure*

• Check that all wells are being washed evenly. • Pipets are fundamentally important to the accuracy of the assay. They should be checked on a

regular bases for precision and accuracy of delivery volumes*. Poor handling of the plates

• Clean the bottom of plates with a kimwipe before inserting into the reader. Film, dirt or fingerprints on the bottom of the plate could interfere with optical density readings resulting in poor CV’s.


• Avoid stacking plates. Keep separated. • Always keep plates sealed with plate covers during incubation steps.

• Always use the same procedure for addition of reagents. Using the wrong plates

• Always use the recommended plate for a particular assay. • Never use a tissue culture grade plate for EIA; it will result in high variability.

Water quality*

• This can be a major problem for the daily functioning of assays within a laboratory as well as for standardization of assays across laboratories even when identical reagents are used.

Laboratory Glassware*

• All glass and plastic should be cleaned and rinsed well in distilled water. This avoids the introduction of contaminants or adverse pH conditions into the assay. An enzymatic type detergent (such as Terg-A-Zyme) should be used to wash dishes.

Timing of steps*

• Individual steps should be timed accurately. For example for a 1-hour incubation step, no more than 5 minutes either way should be tolerated.

Temperature*

• Temperature can have an impact on assay development. Make a note of temperature and its variation during the year, this may explain variation in results at different times.


XIII. SAMPLE COLLECTION/STORAGE METHODS It is very important that all samples be properly labeled with the animal’s name or studbook/identification number, facility, and date. BLOOD:

• When collecting multiple samples per day, label the time. This is especially important for hormone challenges, like ACTH and GnRH.

• After blood has been collected and centrifuged, pour serum into the labeled storage tube. Leave room in the tube for expansion of sample during freezing (e.g., put 4 ml into a 5-ml vial). Place

samples upright into storage box and freeze at -20°C. Store samples in the box from left to right and front to back (see box below). Place the first sample in space #1 and repeat for each new sample added. This way, when processing samples, they are already organized and in order by date and time.

• Remember to keep all samples frozen until analysis.

• Label the outside of the storage box with the name of the animal, species (if necessary), the location of the animal, the matrix (urine, serum, fecal extract), and the dates of the first and last sample in the box. For example: Shanthi, Asian Elephant, National Zoo, urine, 1 Oct 00 - 20 Nov 00.

Sample storage box:

91 95 100

81 85 90

71 75 80

61 65 70

51 55 60

41 45 50

31 35 40

21 25 30

11 15 20

1 2 3 4 5 6 7 8 9 10


URINE:

• Label collection tube with animal’s name, date, and time collected. If necessary, record if the sample may have been contaminated with feces, water, etc. Whenever possible, centrifuge sample and decant supernatant to remove debris.

• Pour urine sample into labeled tube and place in storage box. Leave room in the tube for expansion

of sample during freezing (e.g., put 4 ml into a 5 ml vial). Store sample in freezer at -20°C. Store samples as described previously.

• If storing samples at RT or in the refrigerator (for up to a month) instead of freezing, add 10% ethanol to each tube as a preservative.

• Thoroughly clean syringes or cups used to collect urine samples to prevent cross-contamination. FECES:

• Place feces in collection bag or tube labeled with animal’s name, date, and time (if relevant) and

store at -20°C. • For fecal extracts, label storage tube with animal’s name, date, time, and the extraction medium. For

example: Shanthi, 1 Oct 00, 0800 h, neat extract MeOH.

• If making dilutions from the extract and the dilutions will be stored as well, label the storage tube with the animal’s name, date, time, and the dilution. Remember, dilutions are always made in assay buffer.

• Place fecal extract tubes in storage boxes as described previously and store at

-20°C. Fecal extracts are in alcohol so they will not freeze. FECAL DILUTIONS For fecal extracts and urine samples, dilutions often have to be made, especially for extracts stored in alcohols.

• The appropriate dilution to use is determined from the parallelism curve. • Dilutions are always made with assay buffer. Dilutions are always stated as 1 to a number. For

example: 1:100 (1 + 99) means one part sample to 100 total parts volume.

• Once fecal extract samples have been diluted in buffer, they can be frozen indefinitely until analysis.


XIV. HORMONE EXTRACTION METHODS Definition: Extraction is a method by which components of interest in a sample are isolated, removed or concentrated, making it free of interference from other particulates found within the matrix (fecal, urine, blood or saliva). This generally refers to steroids. For most assay systems, free steroids must be extracted from the sample matrix. A few systems have been developed to analyze unextracted samples (e.g., commercial RIA kits). The extraction method used is dependent on the properties of the component of interest. For more specific extractions (eg. pulling in a specific group of steroids, or removing proteins or lipids from a sample) varying solvents and/or ratios of two or more solvents can be used. BLOOD SERUM OR PLASMA

• Ether extraction is most commonly used. Diethyl ether is used for progestagens; ethyl ether is used for estrogens and androgens. Standard protocol involves extracting with a 10:1 volume of ether: sample by vortexing for 1 minute followed by snap freezing in liquid nitrogen or dry ice/acetone. The ether supernatant is dried and the sample reconstituted in assay buffer, vortexed for 1 min and stored frozen (-20˚C) until analyzed.

URINE

• Ether can be used for gonadal steroids similar to blood serum/plasma extractions. • For corticoids, dichloromethane is very effective. For that method, urine (0.5 ml) is combined with 1

ml dichloromethane in a 12 x 75 mm polypropylene tube, capped tightly, shaken gently for 10 min and centrifuged (1500 x g) for 5 min. The lower dichloromethane fraction is removed by aspiration using a Pasteur pipet and placed into a glass 12 x 75 mm tube. Six hundred �� are then transferred to another glass 12 x 75 mm tube using a positive displacement pipet and dried under air. Samples are reconstituted in assay buffer, vortexed for 1 min and stored frozen (-20˚C) until analyzed.

CONJUGATED STEROID ANALYSIS

• Steroid conjugates (sulfates or glucuronides) can be assayed directly using antibodies that crossreact with conjugates.

• Conjugated steroids can be assessed indirectly after enzyme hydrolysis (e.g., sulfatase, glucuronidase or ‘snail juice’, Helix pomatia, which contains both glucuronidase and sulfatase activity) and assaying the resulting free steroids. Extract urine, serum or fecal extracts (resuspended in 0.5 ml buffer) with 10 volumes of diethyl ether to separate water-soluble from ether-soluble forms. Hydrolyze residual aqueous samples diluted 1:1 in 0.5 ml in 0.1 M acetate buffer (pH 5.0) with 50 µl ß-glucuronidase/aryl sulfatase (20,000 Fishman U/40,000 Roy U, respectively; Boehringer Mannheim Corp., Indianapolis, IN) at 37˚C for 24 hours. Samples are then extracted with 10 volumes of diethyl ether to separate enzyme-hydrolyzable (organic phase) from non-hydrolyzable (aqueous phase) forms.


DRY AND WET WEIGHT FECAL EXTRACTION – BOILING METHOD

1. Feces can be dried using a lyophilizer, savant rotary evaporator or conventional oven (~50˚C to 95˚C oven temps have been reported for several hours or until dry; i.e., weight no longer changes). Solar ovens are not recommended due to potential steroid alteration by UV light. Drying times will vary for each technique and even among individual or species samples.

2. Weigh out feces in numbered 16x125 mm glass tubes. Record weight of each sample.

3. The following weights are suggestions:

Dry: Herbivores Weigh out 0.1 g (+/- 0.01) of dry powdered feces Carnivores Weigh out 0.2 g (+/- 0.02) of dry powdered feces Wet: Herbivores and Carnivores Weigh out 0.5 g (+/- 0.01) of wet feces

4. If monitoring extraction efficiency, add appropriate amount of tracer or unlabeled hormone to each tube

(see below).

5. Add 4.5 ml of ETOH and 0.5 of dH2O (90% ETOH) to each tube and vortex briefly.

6. Boil tubes in boiling water bath (96°) for 20 minutes. Keep rack from touching the bottom to prevent splattering of tube contents. Add 100% ETOH as needed to keep from boiling dry.

7. Bring the volume of the extract up to approximately pre-boil levels with 100% ETOH.

8. Centrifuge the samples at 2500 rpm for 20 minutes, making sure the centrifuge is balanced.

9. Pour off the extracts into a second set of identically labeled 16x125 mm tubes.

10. Add 4.5 ml ETOH and 0.5 ml of dH2O to the original tubes containing the fecal pellets and vortex

each tube for 30 seconds. 11. Centrifuge these tubes at 2500 rpm for 20 minutes. 12. Pour off the extract into the second set of tubes containing the first extract.

13. Dry down the second set of tubes under air in a warm water bath.

14. Re-suspend the dried down extracts in 3 ml ETOH, vortex and dry down under air in a warm water bath.

15. Bring up in 1 ml of MEOH or dilution buffer, vortex briefly, sonicate for 15 minutes.

16. Pour off extracts into labeled 12x75 mm plastic tubes, if using MeOH dry down extract under air.


FECAL EXTRACTION – VORTEXING METHOD

1. Steps 1-4 same as above.

2. Add 4.5 ml of ETOH and 0.5 of dH2O to each tube and vortex briefly.

3. Load samples into a rack shaker. Shake at room temperature for 4 hours or at 4°C overnight (these are suggested times).

4. Vortex for 30 min. Most hormone is extracted in the first 5 min, but some species may take longer.

5. Centrifuge at ~2500 rpm for 20 min.

FECAL EXTRACTION – SHAKING METHOD

1. Steps 1-4 same as above.

2. Load samples onto a multitube vortexer.

3. Vortex each sample at a speed of ~60-70 (fast enough so a good vortex is created; not so fast that the tubes work their way up out of their place), with 1 pulse/sec.

4. Vortex for 30 min. Most of the steroids are probably extracted within 5 min, but samples from some

species may take longer due to consistency differences.

5. Centrifuge at ~2500 rpm for 20 min.

6. Follow Steps 9-16 as above.


EXTRACTION EFFICIENCY PROTOCOL Use of radioactive tracer (most efficient and accurate method)

1. Dilute H3 or C14 labeled hormone to about 2000 dpm/100 �l. 2. Add 100 �l of tracer (H3 or C14) to all tubes. At the same time add 100 �l to two scintillation vials with

same repeater pipet as that used to add tracer to samples. 3. Extract the sample as described previously. 4. Take 100 �l from each sample extract (the 1 ml MeOH extract from step 14 of the extraction protocol)

and add to scintillation vials. Do in singlicate, not duplicate. 5. Add 3 ml of scintillation fluid to all vials, including the two vials with tracer. Create 2 blank vials and

add only 3 ml of scintillation fluid. 6. Cap all vials firmly and invert to mix the fluid and set aside for an hour. 7. Determine DPM in a Beta Counter.

8. Calculations:

• Average your blank values (zero values) and subtract this from everything, including your totals.

• Average totals. • Sample x 10 (because is a tenth of the total sample volume)/averaged total value = multiply by

100 to get a percentage. • Between 80 and 100 is acceptable. If the percent recovery is over 100 the quench needs to be

checked because the color of the extract may be interfering with the counter’s ability to read the sample. If the sample is too dark it needs to be diluted out by adding more scintillation cocktail when you run the next recovery.

Use of unlabeled hormone (requires two extractions per sample)

1. Dilute cold steroid in assay buffer to a concentration ~10 times the value of the 50% binding standard concentration at the �l concentration used in each assay (i.e., 50 �l for most assays). This concentration is an estimate. Because samples are diluted before analysis, the concentration added may need to be adjusted. Goal should be to spike samples with enough steroid so that the value falls within the standard range. Best results will be obtained using ‘low’ concentration samples so that the endogenous concentration falls near the 80% binding portion of the curve.

2. Add cold steroid dilution to all tubes (i.e., 50 �l for most assays) (i.e., spiked sample). Also, set aside

duplicate sample tubes for each sample with no added hormone (to determine endogenous concentration in unspiked sample) (i.e., unspiked sample).

3. Extract samples as describe previously. 4. Dilute samples as determined by parallelism tests and analyze as per assay protocol. 5. Calculations:

• Average the concentration for each paired set of samples (one extracted with added steroid, one extracted with no added steroid).

• Subtract the value of the unspiked sample from the spiked sample and determine the difference. • Calculate the percentage difference for the recovery value (the difference between spiked and

unspiked sample). Use of repeated extraction to determine extraction consistency


1. While not a measure of extraction recovery per se, extracting a large number of duplicates of individual samples can give an indication of how consistent the extraction process is. Thus, even if the actual recovery is unknown, if the extraction consistency is good (<15-20%), the data generated will be okay.

2. This procedure should be done occasionally (~1 trial per month) to ensure continued precision.

3. Take 5 individual dried fecal samples, mix well and split into 20 subaliquots. Extract each subaliquot and calculate the %CV among aliquots within each sample.


Fecal Extraction Sheet

Species: Date: Sex: ID: Zoo:

Sample DATE WEIGHT Sample DATE WEIGHT Sample DATE WEIGHT

1 50 99

2 51 100

3 52 101

4 53 102

5 54 103

6 55 104

7 56 105

8 57 106

9 58 107

10 59 108

11 60 109

12 61 110

13 62 111

14 63 112

15 64 113

16 65 114

17 66 115

18 67 116

19 68 117

20 69 118

21 70 119

22 71 120

23 72 121

24 73 122

25 74 123

26 75 124

27 76 125

28 77 126

29 78 127

30 79 128

31 80 129

32 81 130

33 82 131

34 83 132

35 84 133

36 85 134

37 86 135

38 87 136

39 88 137

40 89 138

41 90 139

42 91 140

43 92 141

44 93 142

45 94 143

46 95 144

47 96 145

48 97 146

49 98 147


XV. CALCULATING EIA RESULTS READING THE PLATE

• Since the product of EIA is color, it can be read in two ways: 1) by eye inspection; or 2) by using a spectrophotometer.

• Reading plates by eye is usually only used for positive/negative type results. • The product of the substrate catalysis by enzyme is measured by transmitting light of a specific

wavelength through the product and measuring the amount of light adsorption by a plate reader (i.e., optical density).

• Since different products can be produced in EIAs, the correct filter for the detection of the wavelength must be used.

CALCULATING RESULTS FROM OPTICAL DENSITY (OD)

• There are no set rules on how to set up a plate; it is a matter of choice. • We run plates by moving down columns (standard curve in duplicate – zeros; standards S2-S10;

controls, C1-C2; samples, T1-T26; standard curve in singlet to account for any time lag in loading the plate).

S= Standard C= Control T= Test Sample 1 2 3 4 5 6 7 8 9 10 11 12

A Blank Zero S5 S9 T1 T5 T9 T13 T17 T21 T25 S5

B Blank Zero S5 S9 T1 T5 T9 T13 T17 T21 T25 S6

C S2 S6 S10 T2 T6 T10 T14 T18 T22 T26 S7

D S2 S6 S10 T2 T6 T10 T14 T18 T22 T26 S8

E S3 S7 C1 T3 T7 T11 T15 T19 T23 Zero S9

F S3 S7 C1 T3 T7 T11 T15 T19 T23 S2 S10

G S4 S8 C2 T4 T8 T12 T16 T20 T24 S3 Zero

H S4 S8 C2 T4 T8 T12 T16 T20 T24 S4 Zero AVERAGE BLANK VALUE:

• This is the average OD of both blank wells (background or non-specific binding) • This average is then removed from every other well on the plate


DATA MATRIX/TABLE : OD

• These are the final OD readings from the plate reader after the background has been removed. • The printout will identify which OD value corresponds to which well.

Example: “Well A3” OD equals 0.722 on print out

1 2 3 4 5 6 7 8 9 10 11 12

A Blank 0.722

B Blank

C

D

E

F

G

H

Results:

• To determine % specific binding, calculate the average OD of the “zero” wells

1 2 3 4 5 6 7 8 9 10 11 12

A .831

B .928

C

D

E .864

F

G .860

H .877


Example: (Well A2, B2, 11E, 12G, 12H)/5 From printout - (0.831 + 0.928 + 0.864 + 0.860 + 0.877)/5 = 0.872

• “0.872” is the denominator by which all other OD values are divided

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E .857

F

G

H Example: OD from “Well E2” equals (0.857/0.872) * 100 % = 98.2%

• Calculate the % binding for each of the standard wells.

1 2 3 4 5 6 7 8 9 10 11 12

A 82 26 81

B 82 25 68

C 105 70 15 52

D 103 68 14 35

E 98 55 24

F 99 52 94 15

G 92 39 93

H 92 38 94

• Take the average % binding of the standards that have been run in triplicate. Example: ‘Standard 10’ which is 1000 pg/well is found in wells C4, D4 and F12

From print out – (15 + 14 + 16)/3 = 15%


• Now plot the standard curve.

ŹŹŹŹŹ

0

20

40

60

80

100

120

1 10 100 1000 10000

Standard Concentration (logrithmic scale)ŹŹŹŹŹŹ

%B/TB

• Calculate the % binding of all controls and test samples

1 2 3 4 5 6 7 8 9 10 11 12

A 79 80 73 68 67 70 121

B 74 78 64 59 63 63 112

C 75 79 71 71 65 108 115

D 76 78 71 69 63 109 108

E 44 77 76 73 64 65 106

F 43 73 73 71 56 62 106

G 81 78 70 40 69 76 108

H 81 77 67 43 69 76 109


• Calculate dose for each control and test sample by extrapolating data from standard curve

ŹŹŹŹ

0

20

40

60

80

100

120

1 10 100 1000 10000

Standard Concentration (logrithmic scale)ŹŹŹŹŹŹŹŹ

% B/TB

1st

2nd

Example:

• Test sample one or “T1” was placed into “Well A5” and “Well A2”. • The percent binding was calculated to be 79% and 74%, respectively.

• The average % binding would be 76%. • The concentration at 76% is 42.85 pg/well. • Results are converted to concentration/ml of sample.

Example:

• 42.85 pg/well is equal to 42.85 pg/50 µl • The 50 is much was loaded into the well, for the cortisol assay this is 50 µl/well.

Thus, 42.85 pg/50 µl = 0.857 pg/µl

0.857 pg/µl * 1000 (1 ml) = 857 pg/ml 857 pg/ml/1000 (pg to ng) = 0.857 ng/ml

CONVERTING DATA FROM FECES Example:

• Need weight of feces originally weighed out for extraction (e.g., 0.5 g) • Final volume from the extraction (e.g., 1 ml)

• From the assay calculate the mass (e.g., pg/well to ng/ml….say 120 ng/ml) • Dilution factors need to be calculated into the data (e.g., samples ran at a 1:10 – then 120 * 10 =

1200 ng/ml) Thus, 1200 ng/0.5 g = x ng/1g of feces = 240 ng/g of feces


CONVERTING DATA FROM URINE Example:

• From the assay calculate the mass (e.g., pg/well to ng/ml….e.g., 20 ng/ml) • Dilution factors need to be calculated into the data (e.g., samples ran at a 1:10 – then 10 * 20 = 200

ng/ml)

• Then divide the sample value by the creatinine value (e.g., 0.182 mg/ml creatinine) Thus, 200 ng/ml divided by 0.182 mg/ml = 1098.9 ng/mg creatinine

TIPS FOR READING EIA PLATES

• Clean bottom of plate before reading • Make sure the right filter is used to read the plate

• Get print out of OD’s • Remove average background OD’s from all other OD values • Calculate average ‘zero’ OD value

• Use this to calculate % binding of all other wells - (OD of each well/ ‘zero’ OD average value) *100% • Plot the standard curve – concentration (x axis - log scale) vs % binding (y axis - linear scale)

• Average % binding’s for each sample (controls and test samples) • Extrapolate concentrations based on % binding

• Convert data to a per ml or per gram mass concentration (Example: pg/well to ng/ml)


XVI. ASSAY PROTOCOLS Note: Throughout the Assay Protocol section, reference to “room temperature” means 65 - 80°F (22 - 26°C). Referral to “freezing” of samples means -4°F (-20°C) or colder. CREATININE ASSAY Definition: Creatinine is a breakdown product of muscle protein that is excreted in urine at a constant rate. It is a way to measure how concentrated a urine sample is and whether or not a certain sample is viable. For instance, if it contains too much water and thus mass within the sample is too dilute to measure, it is not a viable sample.

• Thaw all samples to be analyzed.

• Creatinine can degrade so samples should be thawed once or kept cold. It is best to run the creatinine assay after the first thaw of the sample, but as long as the sample is kept cold after thawing it can be refrozen and run at a later time.

PROTOCOL FOR CREATININE ASSAY - per plate

1. STANDARDS

• Standard values used are 100, 50, 25, 12.5, and 6.25 µg/ml • Dilute top standard stock (Sigma – 925-11; 10 mg/dl or 100 µg/ml, 4˚C) serially 2-fold using 200 µl

stock plus 200 µl assay buffer or tap water and mix well

• Use same buffer or tap water as the zero standard (Note: water quality is not critical) 2. SAMPLES

• Dilute samples to appropriate amount (1:5 - 1:100 in buffer or water) 3. PLATE LOADING

• Use Dynatech plates (Immulon 4 Flat Bottom Plates – Cat # 0110103855) • Add 50 µl per well of standard and sample in duplicate according to plate map

• Speed of addition is unimportant as this is not a binding assay • Start at A1 and go down each set of columns • Pipet all solutions in this order

• Avoid splashing or touching the samples in the wells • Add 50 µl of tap water to each well using the repeater pipet

• Add 50 µl per well 0.75 N NaOH • Add 50 µl per well 0.4 N picric acid using the repeater pipet

• Shake plate briefly to mix (by tapping) and incubate at RT for 30 minutes 4. PLATE READING

• Wipe bottom of plate to make sure it is clean • Read at 490 nm

5. ASSAY DILUTION/VALUE CUTOFFS

• Avoid running samples at <1:5 or >1:100 dilution

• Any sample with a creatinine value of <0.1 mg/ml is too dilute and should not be used


Creatinine Assay Template

ASSAY SPECIES

DATE ANIMAL ID

DILUTION

1 2 3 4 5 6 7 8 9 10 11 12

A 0 50

B 0 50

C 6.25 100

D 6.25 100

E 12.5

F 12.5

G 25

H 25


CHECKERBOARD TITRATION FOR EIA Definition: A checkerboard titration is used to determine the appropriate concentrations of enzyme conjugate tracer and first antibody to use in an EIA. Different concentrations of antibody are run horizontally, whereas tracer dilutions are run vertically along the plate (see template). A high and ‘zero’ standard is run at each level. The ideal concentration of antibody and conjugate is one that provides good distinction between high and ‘zero’ standards, at the highest dilutions possible. The following is an example of a Checkerboard Titration for the EC R522-2 assay. Each assay will require testing different concentrations of reagents. Follow these steps along with the titration plate map: Day 1:

• Prepare a 1:100 dilution of the EC R522-2 Abº by adding 20 µl of stock to 2 ml of coating buffer, label this tube “Working Abº”

• You will now prepare several Abº concentrations. The directions are as follows: � Prepare a 1:15,000 by adding 13.33 µl of Working Abº to 1986.7 µl Coating Buffer (you can

do this precisely by adding 2 ml of coating buffer to a tube, remove 13.33 µl of the coating buffer with a 100 µl pipet, and using the same pipette tip take out 13.33µl of the 1:100 Working Abº and add it to the tube containing the buffer. This step can be followed for the following Abº dilutions and the HRP dilutions in step 9, just adjust the amounts accordingly).

� Prepare a 1:20,000 by adding 10 µl of working Abº to 1990 µl of Coating Buffer. � Prepare a 1:30,000 by adding 10 µl of working Abº to 2990 µl of Coating Buffer. � Prepare a 1:40,000 by adding 10 µl of working Abº to 3990 µl of Coating Buffer. � Prepare a 1:50,000 by adding 10 µl of working Abº to 4990 µl of Coating Buffer.

• In this step you will coat your plate with the Abº dilutions you made in step 2 according to the plate map:

� Coat columns 1 & 2 with 50 µl of the 1:15,000 dilution in each well. � Coat columns 3 & 4 with 50 µl of the 1:20,000 dilution in each well. � Coat columns 5 & 6 with 50 µl of the 1:30,000 dilution in each well. � Coat columns 7 & 8 with 50 µl of the 1:40,000 dilution in each well. � Coat columns 9 & 10 with 50 µl of the 1:50,000 dilution in each well. � For columns 11 & 12 add 50 µl of Coating buffer to each well (do not add Abº to columns 11

& 12). Cover with acetate plate sealer and leave overnight at 4ºC.

Day 2:

• Wash the plate as usual.

• Add 50 µl EIA buffer to all wells of the plate, cover with plate sealer, and let plate set at room temperature for 2 - 5 hours.

• You will need at least 3mls of top standard (200 pg/well, see “EC (EC R522-2) Stock Preparations” of the EC R522-2 protocol on how to make this up) and EIA buffer for the plate (you will not need to dilute the standard).

• For this procedure it is best to prepare a fresh batch of HRP working stock. Add 20 µl of HRP stock to 980 µl EIA buffer.

• Prepare several HRP concentrations to test out. The directions are as follows (keep all solutions cool while: � For the 1:10,000 add 20 µl of HRP working stock to 1980 µl of EIA buffer. � For the 1:15,000 add 13.33 µl of HRP working stock to 1986.7 µl of EIA buffer. � For the 1:20,000 add 10 µl of HRP working stock to 1990 µl of EIA buffer. � For the 1:25,000 add 12 µl of HRP working stock 2988 µl of EIA buffer.

Loading the Plate:


• Add 20 µl of EIA buffer to each well in rows A, C, E, and G.

• Add 20 µl of top standard (200 pg/well) each well in to rows B, D, F, and H.

• Add the respective HRP’s to the plate (following the plate map): � 50 µl of HRP 1:10,000 to each well in rows A & B. � 50 µl of HRP 1:15,000 to each well in rows C & D. � 50 µl of HRP 1:20,000 to each well in rows E & F. � 50 µl of HRP 1:25,000 to each well in rows G & H.

• Let plate incubate for two hours at room temp.

• Wash plate and add substrate as you usually do when running the EC assay.

• After 30 minutes read the plate. Interpreting the results:

1. Ideal Abº and HRP concentrations are those that produce OD’s of the zeros in rows A, C, E, and G between 0.8 and 1.0.

2. Average all OD’s for the 0’s and Top Standards for each Abº and HRP dilution set. For example: average the 0’s that are in wells A1 and A2 and the top standards in B1 and B2 (A1, A2, B1 and B2 make up the block of wells that are included in the Abº dilution of 1:15,000 and HRP dilution of 1:10,000).

3. Divide the top standard average of a dilution set by the 0 average of the same dilution set to get the displacement number. Calculate the displacement number for all the Abº and HRP dilution sets that have 0’s with OD’s between 0.8 and 1.0.

4. The Abº and HRP dilution set with the highest displacement number is the Abº and HRP dilution set to use for the assay.


Checkerboard Titration Plate Map

Assay Species

Date Animal ID

Ab CONCENTRATIONS

1:15,000 1:20,000 1:30,000 1:40,000 1:50,000 0

1 2 3 4 5 6 7 8 9 10 11 12

0 A

Hig

h

Sta

ndard

B

1:1

0,0

00

0 C

Hig

h

Sta

ndard

D

1:1

5,0

00

0 E

Hig

h

Sta

ndard

F

1:2

0,0

00

0 G

Hig

h

Sta

ndard

H

1:2

5,0

00

HR

P C

ON

CE

NT

RA

TIO

NS


CORTISOL EIA Day 1: 1. Plate coating

• Use NUNC Maxisorb plates

• Add 50 µl antibody stock ( • 1:85, -20ºC) to 5 ml coating buffer (working dilution 1:8500)

• Add 50 µl per well of antibody solution to plate. • do not coat column 1 - start at A2 and go down each column (see plate map).

• Pipet all solutions in this order.

• Tap plates gently to ensure that coating solution covers bottom of well • Label, cover with acetate plate sealer and leave overnight (no less than 12 hrs) at 4ºC.

Day 2: 2. Standards

• Standard values used are: 1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8 and 3.9 pg/well.

• Dilute standard working stock (20 ng/ml or 1000 pg/well) serially (2-fold) by using 250 µl stock plus 250 µl EIA buffer.

3.Samples/controls

• Dilute urine or fecal samples in dilution buffer to appropriate dilution. • Prepare High and Low control

4. HRP

• Cortisol HRP working dilution is 1:20,000. • Add 25 µl of HRP working stock to 5 ml EIA buffer to make the working dilution (keep this

solution cool) 5. Plate washing

• Wash the plate five times with wash solution. • Blot the plate on paper towel to remove excess wash solution.

6. Plate loading

• Pipet 50 µl of standard, control and samples per well as quickly and accurately as possible, according to plate map.

• Add 50 µl of diluted cortisol HRP (step 4) to all wells that contain standard, control, or sample. Avoid splashing.

• No more than 10 minutes should pass during this process

• Cover plates with acetate plate sealer and incubate at room temperature for exactly 1 hour.

7. Plate washing


• Plates are fairly stable at this point and can be left upside down on bench top until all plates are washed (no more than 20 minutes)


8. Substrate

• Prepare ABTS substrate immediately before use (within 20 min). • Combine 40 µl 0.5 M H2O2, 125 µl 40 mM ABTS and 12.5 ml substrate buffer, and mix

well.

• Add 100 µl ABTS substrate to all wells that contain standard, control, or sample. • Cover with plate sealer and incubate at room temperature with shaking. • Plate color development will vary based on age of HRP and/or Ab, but should be no

greater than one hour. 9. Plate reading

• Optical density (OD) of 0 wells should read 1 or less.

• Optimal readings for 0 wells: > 0.7 to < 1 OD. • Read at 405 nm (reference 540 nm).

CORTISOL STOCK PREPARATIONS – per plate 1. Antibody

• Dilute cortisol R4866 at a dilution of 1:85 by adding 24 µl of stock to 2 ml of coating buffer.

• Aliquot 300-400 µl into O-ring vials and store at -20ºC.

• Store antibody stock at -80ºC. 2. HRP Conjugate

• Dilute cortisol-horseradish peroxidase (HRP) 1:100 by adding 25 µl of stock to 2.475 ml EIA buffer for a working stock and store at 4ºC.

• Store HRP stock at -80ºC. 3. Standards

• Weigh out 1 mg cortisol (Sigma Diagnostics) and add to 1 ml ETOH for a 1 mg/ml primary stock.

• Dilute 1 mg/ml primary stock 1:100 by adding 100 µl to 10 ml ETOH for a 10 µg/ml secondary stock.

• Dilute 10 µg/ml secondary stock 1:500 by adding 100 µl to 49.9 ml of EIA Buffer for a 20 ng/ml (1000 pg/well*) working stock.

• Aliquot working stock and store all stocks at -20ºC. * a well is equal to 50 µl, the amount used in the assay.

4. Controls

• Use urine or extracted fecal samples with a high corticoid levels to make controls.

• Make a pool of high corticoid level urine or extracted feces (~20 ml). • Serially dilute pool and run on assay.

• Find the dilutions that bind at ~70% and ~30%. • Use the pool to make up two separate stocks for low and high controls using the

dilutions that bound at 70% and 30% respectively.

• Make up enough controls to run on at least 500 assays (for this you may need more than the 20 ml used for the pool. Any species will do so long at the urine has high corticoid levels.)


CORTISOL EIA Template

ASSAY SPECIES

DATE ANIMAL ID

DILUTION

1 2 3 4 5 6 7 8 9 10 11 12

A

Blank 0 31.2 500 31.2

B

62.5

C

3.9 62.5 1000 125

D

250

E

7.8 125 C1 0 500

F

3.9 1000

G

15.6 250 C2 7.8 0

H

15.6 0


PREGNANE (CL425) EIA Day1:

1. Plate Coating • Use NUNC Maxisorb plates.

• Add 25 µl antibody stock (1:50, -20ºC) to 5 ml coating buffer (working dilution 1:10,000). • Add 50 µl per well of antibody solution to plate. • do not coat column 1 - start at A2 and go down each column (see plate map).

• Pipet all solutions in this order. • Tap plates gently to ensure that coating solution covers bottom of well.

• Label, cover with acetate plate sealer and leave overnight (no less than 12 hrs) at 4ºC. Day 2:

2. Standards

• Standard values used are: 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 pg/well. • Dilute standard working stock (4 ng/ml or 200 pg/well) serially (2-fold) by using 250 µl

stock plus 250 µl EIA buffer. 3. Samples/controls

• Dilute urine or fecal samples in dilution buffer to appropriate dilution.

• Prepare High and Low controls. 4. HRP

• Pregnane HRP working dilution is 1:40,000.

• Add 30 µl of HRP working stock to 6 ml EIA buffer to make the working dilution (keep this solution cool).

5. Plate washing


6. Plate loading


• Add 50 µl of diluted Pregnane HRP (step 4) to all wells that contain standard, control, or sample. Avoid splashing.

• No more than 10 minutes should pass during this process • Cover plates with acetate plate sealer and incubate at room temperature for exactly 2

hours. 7. Plate washing

• Wash the plate five times with wash solution.

• Blot the plate on paper towel to remove excess wash solution. • Plates are fairly stable at this point and can be left upside down on bench top until all

plates are washed (no more than 1 hour).


8. Substrate


well.





PREGNANE STOCK PREPARATION – per plate 1. Antibody

• Dilute monoclonal CL425 at a dilution of 1:50 by adding 100 µl of stock to 4.9 ml of coating buffer.


• Store Antibody stock at -80ºC. 2. HRP Conjugate

• Dilute progesterone-3CMO-horseradish peroxidase 1:200 by adding 25 µl of stock to 4.975 ml EIA buffer for a working stock and store at 4ºC.


• Weigh out 0.5 mg progesterone (Sigma Diagnostics Cat. # P 0130) and add to 5 ml ETOH in a scintillation vial for a 100 µg/ml primary stock solution

• Dilute 100 µg/ml (100 000 ng/ml) primary stock 1:100 by adding 40 µl to 4 ml ETOH for a 100 ng/ml secondary stock.

• Dilute 100 ng/ml secondary stock 1:2500 by adding 200 µl to 49.8 ml of EIA Buffer for a 4 ng/ml (200 pg/well*) working stock.

• Aliquot working stock and store all stocks at -20ºC. *a well is equal to 50 µl, the amount used in the assay.

4. Controls

• Use urine or extracted fecal samples with a high progestin levels to make controls.

• Make a pool of high progestin level urine or extracted feces (~20 ml). • Serially dilute pool and run on assay. • Find the dilutions that bind at ~70% and ~30%.

• Use the pool to make up two separate stocks for low and high controls using the dilutions that bound at 70% and 30% respectively.

• Make up enough controls to run on at least 500 assays (for this you may need more than the 20 ml used for the pool. Any species will do so long at the urine has high progestin levels).


PREGNANE EIA Template

DATE ANIMAL IDDILUTION

1 2 3 4 5 6 7 8 9 10 11 12

ABlank 0 6.25 100 6.25

B12.5

C0.78 12.5 200 25

D50

E1.56 25 C1 0 100

F0.78 200

G3.12 50 C2 1.56 0

H3.12 0


TESTOSTERONE EIA Day1:

1. Plate Coating

• Use NUNC Maxisorb plates

• Add 66.7 µl antibody stock (1:100, -20ºC) to 5 ml coating buffer (working dilution 1:7500) • Add 50 µl per well of antibody solution to plate. • do not coat column 1 - start at A2 and go down each column (see plate map).

• Pipet all solutions in this order. • Tap plates gently to ensure that coating solution covers bottom of well


2. Standards

• Standard values used are: 600, 300, 150, 75, 37.5, 18.8, 9.4, 4.7 and 2.3 pg/well • Dilute standard working stock (12,000 pg/ml or 600 pg/well) serially (2-fold) by using 250

µl stock plus 250 µl EIA buffer. 3. Samples/controls

• Dilute urine or fecal samples in dilution buffer to appropriate dilution.

• Prepare High and Low controls 4. HRP

• Testosterone HRP working dilution is 1:15,000.

• Add 33.3 µl of HRP working stock to 5 ml EIA buffer to make the working dilution (keep this solution cool).

5. Plate washing


6. Plate loading


• Add 50 µl of diluted testosterone HRP (step 4) to all wells that contain standard, control, or sample. Avoid splashing.

• No more than 10 minutes should pass during this process. • Cover plates with acetate plate sealer and incubate at room temperature for exactly 2

hours. 7. Plate washing

• Wash the plate five times with wash solution.

• Blot the plate on paper towel to remove excess wash solution. • Plates are fairly stable at this point and can be left upside down on bench top until all

plates are washed (no more than 20 minutes).


8. Substrate


well.


greater than one hour.

9. Plate reading



TESTOSTERONE STOCK PREPARATION – per plate 1. Antibody

• Dilute Polyclonal anti-testosterone R156/7 at a dilution of 1:100 by adding 20 µl of stock to 2 ml of coating buffer.

• Aliquot 300-400 µl into O-ring vials and store at -20ºC. • Store antibody stock at -80ºC.

2. HRP Conjugate

• Dilute testosterone-horseradish peroxidase (HRP) 1:100 by adding 25 µl of stock to 2.475 ml EIA buffer for a working stock and store at 4ºC.


• Weigh out 1 mg 17-hydroxy-4-androsten-3-one (Steraloids Cat. #A6950) and add to 1 ml ETOH for a 1 mg/ml primary stock.

• Dilute 1 mg/ml primary stock 1:100 by adding 100 µl to 10 ml ETOH for a 10 µg/ml secondary stock.

• Dilute 10 µg/ml (10,000 ng/ml) secondary stock 1:100 by adding 250 µl to 24.75 ml EIA Buffer for a 100 ng/ml tertiary stock.

• Dilute 100 ng/ml tertiary stock 1:8.33 by adding 12 ml to 88 ml of EIA Buffer for a 12,000 pg/ml (600 pg/well*) working stock.


4. Controls

• Use urine or extracted fecal samples with a high testosterone level to make controls.

• Make a pool of high testosterone level urine or extracted feces (~20ml). • Serially dilute pool and run on assay. • Find the dilutions that bind at ~70% and ~30%.


• Make up enough controls to run on at least 500 assays (for this you may need more than the 20ml used for the pool. Any species will do so long at the urine has high testosterone levels.)

TESTOSTERONE EIA Template


ASSAY SPECIES

DATE ANIMAL ID

DILUTION

1 2 3 4 5 6 7 8 9 10 11 12

ABlank 0 18.8 300 18.8

B37.5

C2.3 37.5 600 75

D150

E4.7 75 C1 0 300

F2.3 600

G9.4 150 C2 4.7 0

H9.4 0


ESTRONE CONJUGATE (EC R522-2) EIA Day 1:

1. Plate Coating

• Use NUNC Maxisorb plates.

• Add 12.5 µl antibody stock (1:100, -20ºC) to 5 ml coating buffer (working dilution 1:40,000).

• Add 50 µl per well of antibody solution to plate. • do not coat column 1 - start at A2 and go down each column (see plate map).

• Pipet all solutions in this order. • Tap plates gently to ensure that coating solution covers bottom of well.


2. Plate washing

• Wash the plate five times with wash solution. • Blot the plate on paper towel to remove excess wash solution. • do not allow coated plates to dry - immediately add 50 µl EIA assay buffer per well.

• Incubate plate at RT for 2 - 5 hours. 3. Standards

• Standard values used are: 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 pg EC/well.

• Dilute standard working stock (200 pg/well or 10 ng/ml) serially (2-fold) by using 200 µl stock plus 200 µl EIA buffer.

4. Samples/controls

• Dilute urine or fecal samples in dilution buffer to appropriate dilution. • Prepare High and Low controls

5. HRP • EC-HRP working dilution is 1:25,000.

• Add 20 µl of HRP working stock to 5 ml EIA buffer to make the working dilution (keep this solution cool).

6. Plate loading


• Add 50 µl of diluted EC HRP (step 5) to all wells that contain standard, control, or sample. Avoid splashing.

• No more than 10 minutes should pass during this process.

• Cover plates with acetate plate sealer and incubate at room temperature for exactly 2 hours.

7. Plate washing


• Plates are fairly stable at this point and can be left upside down on bench top until all plates are washed (no more than 1 hour).


8. Substrate


well





EC (EC R522-2) STOCK PREPARATION – per plate

1. Antibody

• Dilute polyclonal anti-EC R522-2 at a dilution of 1:100 by adding 20 µl of stock to 2 ml of coating buffer.


• Store antibody stock at -80ºC. 2. HRP Conjugate

• Dilute estrone-glucuronide-horseradish peroxidase 1:100 by adding 50 µl of stock to 4.95 ml EIA assay buffer for a working stock and store at 4ºC.


• Weigh out 1 mg estrone-glucuronide (Sigma Cat. #E 1752) and add to 100 ml ETOH for a 10 µg/ml primary stock solution.

• Dilute 10 µg/ml primary stock 1:1,000 by adding 100 µl to 100 ml EIA buffer for a 10 ng/ml (200 pg/well*) working stock.


4. Controls

• Use urine or extracted fecal samples with a high estrogen level to make controls. (samples collected during estrus are best).

• Make a pool of high estrogen level urine or extracted feces (~20 ml).

• Serially dilute pool and run on assay. • Find the dilutions that bind at ~70% and ~30%.


• Make up enough controls to run on at least 500 assays (for this you may need more than the 20 ml used for the pool. Any species will do so long at the urine has high estrogen levels).


EC (R522-2) EIA Template

ASSAY SPECIESDATE ANIMAL ID

DILUTION

1 2 3 4 5 6 7 8 9 10 11 12

ABlank 0 6.25 100 6.25

B12.5

C0.78 12.5 200 25

D50

E1.56 25 C1 0 100

F0.78 200

G3.12 50 C2 1.56 0

H3.12 0


XVII. ASSAY REAGENTS AND SUPPLIES 1. STEROID EIA ASSAY RECIPES (full and half recipes) COATING BUFFER Full Half

Na2CO3 (Anhydrous) 1.59 g 0.795 g (Sigma, S2127) NaHCO3 2.93 g 1.465 g (Sigma, S8875) H2O 1000 ml 500 ml pH to 9.6, store at 4˚C STOCK A Stock A 0.2M NaH2PO4 27.8 g 13.9 g (Sigma, S9638) H2O 1000 ml 500 ml STOCK B Stock B 0.2M Na2HPO4 28.4 g 14.2 g (Sigma, S0876) H2O 1000 ml 500 ml ASSAY BUFFER (EIA BUFFER) Stock A 195 ml 97.5 ml Stock B 305 ml 152.5 ml NaCl 8.7 g 4.35 g (Sigma, S9625) BSA 1.0 g 0.5 g (Sigma, A7906) H2O 500 ml 250 ml pH to 7.0, store at 4

oC

“Dilution buffer” for fecal extraction is EIA buffer minus the addition of BSA. WASH SOLUTION CONCENTRATE

NaCl 87.66 g 43.83 g (Sigma, S9625) Tween 20 5.0 ml 2.5 ml (Sigma, P1379) H2O 1000 ml 500 ml store at 4˚C Dilute 10-fold for working wash soln. (i.e.100 ml wash concentrate plus 900 ml H2O), store at RT SUBSTRATE BUFFER

Citric acid (anhydrous) 9.61 g 4.805 g (Sigma, C0759) H2O 1000 ml 500 ml pH to 4.0, store at 4˚C ABTS (40 mM) ABTS 0.55 g (Sigma, A1888) H2O 25 ml pH to 6.0. ABTS is light sensitive - use brown glass or foil for storage store at 4

oC

HYDROGEN PEROXIDE (0.5M) H2O2 (30% Solution) 500 µl (Sigma, H1009) H2O 8 ml

store at 4˚C 2. CREATININE ASSAY RECIPE NaOH (0.75 N)

NaOH Pellets 3.0 g H2O 100 ml This is a strong base so keep in a glass bottle Picric Acid (0.4 M)


Saturated Picric acid 100 ml S925-40 H2O 900 ml OR…. Crystallized Picric acid in H2O 5 g S4255 H2O 500 ml Safety Precautions Picric Acid - Saturated Solution

• Store tightly capped - explosive when dried

• Store in a flame proof cabinet • Poisonous - avoid contact with eyes and mouth • Irritant - avoid contact with skin - wear gloves when handling

• Dilute 1:100 in dH2O for working solution • Flush with water for 5 minutes if it contacts skin or eyes

• Flush with water for 5 minutes when disposing diluted solution down the drain • Incompatible with metals, oxidizing and reducing agents, strong bases, ammonia, concrete and

plaster


Assay Reagents for Steroid enzymeimmunoassays:

Na2CO3 Sigma, S-2127 FW=106.0 Anhydrous 1kg

NaHCO3 Sigma, S-8875 FW=84.01 1kg

NaH2PO4 Sigma, S-9638 MW=138.00 Monobasic 1kg

Na2HPO4 Sigma, S-0876 MW=142.00 Dibasic 1kg

NaCl Enzyme Grade

BSA Sigma, A-7906 Fraction V 100g

Tween 20 Sigma, P-1379 500ml

Citric acid Sigma, C-0759 FW=192.1 Anhydrous 500g

ABTS Sigma, A-1888 5g

H2O2 Sigma, H-1009 FW=34.01 30% w/w solution 500ml

Tris (Trizma base) Sigma, T-1503 FW=121.1 Reagent Grade 1kg

Sodium azide Sigma, S-8032

Tween 80 Sigma, P-1754 500ml

Phosphate citrate buffer (with sodium perborate capsules)

Sigma, P-4922 Good for 100-500 plates.

100 capsules

TMB Sigma T-3405 Good for 50 plates 100 tablets H2SO4 FW=98.08

Reagent grade Make a 0.6M solution

HCL Needed for pH Reagent grade NaOH pellets Sigma, S-5881 1) pH

2) Crt assay

1) 2) need 0.75M (3.0g in 100ml H2O)

Anti-mouse IgG Sigma M8645 whole molecule purified

Good for 20 plates 1mg

Streptavidin Roche Diagnostics 1089153

Boehringer Will last ~ 6 months 500U

Picric acid 1) Saturated 2) Crystalized

in H20

1) Sigma, 925-40

2) Sigma, C-4255

End up with 0.4M solution

1) Dilute saturated stock 1:100 in H2O

2) Weigh out 5g (using plastic) into 500ml H20

CAUTION: Explosive when dried/ poisonous /irritant

Creatinine Standards Sigma, 925-11 Ethyl Alcohol Dehydrated 200

proof For fecal extraction – use ~ 20ml/extraction

Methanol Reagent grade For fecal extraction – use 1ml/extraction


Supplies

Supplier 1 HX 24870A Daigger Nunc mircotitre 96 flat well plates Maxisorp Supplier 2 12-565-135 Fisher Scientific

Mircotitre plates and Sealers Mircotitre plate sealer 3501 Dynex Laboratories

12 x 75 mm glass culture tubes

14 961 26 Fisher Scientific Small Glass Tubes and Racks

Rack - 72 holes, holds 10-13mm

For running EIA’s

60917-500 VWR

16 x 125 mm glass culture tubes

14-961-30 Fisher Scientific Large Glass Tubes and Racks

Rack - 72 holes, holds 16mm tubes

For boiling fecal extracts

Clear 12 x 75 mm plastic tubes

62-526-003 Sarstedt Inc. Small Plastic Tubes Purple 12 mm stopper

caps

To store neat urine and fecal extracts

65-809-003 Sarstedt Inc.

Conical tubes (50 ml) blue caps

2650 Perfector Scientific Large Plastic tubes and/or baggies

ziplock bags with white write-on labels

To store fecal samples

O-ring vials

To store antibody Sarstedt Inc.

Daigger cardboard storage boxes and lids, 3"

LX2860BX Daigger Freezer Boxes and grids

Daigger cardboard storage box grids (100 cell)

Store for urine and extracted fecal samples

LX2860EX Daigger

Plastic wash bottles

500 ml For washing plates and rinsing glassware

Need 3


EQUIPMENT

Pipette (Brinkman Eppendorf 20)

05-402-87 Fisher Scientific







200ul tips 1670-Y Perfector Scientific

Pipettes and Tips

1000ul tips Repeater (Brinkman Eppendorf)

21-380-8 Fisher Scientific Repeater and Tips

Repeater Fisher brand Dispenser Tips

2.5ml, 5ml, 12.5ml, 25ml, 50ml

Vortex Need 3-4 PH meter Calibrators (pH 4, 7, 10),

rinse and holding rack

Sonicator For fecal extractions Magnetic mixer

And magnets

Glass Bottles

1 litre To hold buffers Need 10

Plate Shaker

Should hold up to 4 plates at a time

Boiling H20 bath

For fecal extraction

Drying apparatus

Multitube manifold To dry down fecal extracts

Centrifuge To spin 16 x 125 mm tubes for fecal extraction

Rubber mallet

To pulverize dried fecal samples

Need 3-4

Scale One to weigh out fecals and One to weigh out chemicals

Both Sensitivity of 0.000g

Must have 450nm and 405nm filters; reference filter 540nm or above

Extra Filters and lamps Manuel Disks for revelation software

Plate Reader and Printer

Printer


OTHER……

Timer Sharpie Markers

Kimwipes Parafilm Calculator Glass Scintillation vials

Clipboards Protocols and Assay sheets

Hard and disk copies Covers

Colored Tape

Good H20 supply

Milli-Q, distilled, deionized or reverse osmosis

Critical element

Adaptors and voltage converts for equipment

Plate Reader Printer


XVIII. REVIEW OF STEROID METABOLISM

Adapted from: Th. Steimer, Division of Clinical Psychopharmacology, University Institute of Psychiatry,2, ch. du Petit Bel-Air, 1225 Chêne-Bourg, Geneva, Switzerland

Introduction

Steroids are lipophilic, low-molecular weight compounds derived from cholesterol that play a number of important physiological roles. The steroid hormones are synthesized mainly by endocrine glands such as the gonads (testis and ovary), the adrenals and, during gestation, by the fetoplacental unit, and are then released into the blood circulation. They act both on peripheral target tissues and the central nervous system (CNS). An important function of steroid hormones is to coordinate physiological and behavioral responses for specific biological purposes, like reproduction. Thus, gonadal steroids influence the sexual differentiation of the genitalia and of the brain, determine secondary sexual characteristics during development and sexual maturation, contribute to the maintenance of their functional state in adulthood and control or modulate sexual behavior.

Despite their relatively simple chemical structure, steroids occur in a variety of biologically active forms. This is due in part to the fact that circulating steroids are extensively metabolized by the liver and in target tissues where conversion to an active form is sometimes required. Steroid metabolism is therefore important not only for the production of these hormones, but also for the regulation of their cellular and physiological actions.

Steroid hormones: Structure, nomenclature and classification

The parent compound from which all steroids are derived is cholesterol. As shown in Fig. 1a, cholesterol is made up of three hexagonal carbon rings (A,B,C) and a pentagonal carbon ring (D) to which a side-chain (carbons 20-27) is attached (at position 17 of the polycyclic hydrocarbon). Two angular methyl groups are also found at position 18 and 19. Removal of part of the side-chain gives rise to C21-compounds termed pregnanes (progestins and corticosteroids). Total removal produces C19-steroids, androstanes (including the androgens), whereas loss of the 19-methyl group (usually after conversion of the A-ring to a phenolic structure, hence the term "aromatization") yields the estranes, to which estrogens belong. Individual compounds are characterized by the presence or absence of specific functional groups (mainly hydroxy, keto(oxo) and aldehydes groups) on the carbon skeleton.

Given that at most positions, the functional groups can be oriented either in equatorial or axial position (see Fig. 1b), this type of structure gives rise to a great number of possible stereoisomers (i.e., molecules having the same chemical formula, but a different three-dimensional conformation). Stereoisomerism is very important for biological activity (i.e., for steroid-protein interactions). Substituent groups above the plane of the molecule are said to be in the "ß" position, whereas those situated under the plane of the molecule are said to be in the "�" position. Double-bonds are indicated by the suffix -ene. A complete description of a steroid molecule must therefore include the name of the parent compound (pregnane, androstane or estrane series), and the name, number, position and orientation (� or ß) of all functional groups. Commonly occurring steroids are usually identified by a "trivial" name (e.g., cortisol, testosterone, etc.). Thus, testosterone (trivial name) becomes "17ß-hydroxy-androst-4-ene-3-one".

Steroid hormones can be grouped in various classes according to a number of criteria. Based on their chemical structure they can belong to one of the classes (series) mentioned above (e.g., "a pregnane derivative"). If their site of production is considered to be more important, one can distinguish for example between "ovarian" or "adrenal" steroids. If their biological function is essential, terms like "a glucocorticoid" or "sex steroids" can be used. Finally, classification can also be based on their molecular actions ("an estrogen-receptor agonist") or biochemical effects.

Steroid hormone biosynthesis A general outline of the major biosynthetic pathways

The adrenals produce both androgens and corticosteroids (mineralo- and glucocorticoids), the ovaries (depending on the stage of the ovarian cycle) can secrete estrogens and progestins, and the testis mainly androgens. However, the biochemical pathways involved are strikingly similar in all tissues, the difference in


secretory capacity being mostly due to the presence or absence of specific enzymes. It is therefore possible to give a general outline of the major biosynthetic pathways which is applicable to all steroid-secreting glands, as shown in Fig. 2.

From acetate to cholesterol

Cholesterol can be synthesized in all steroid-producing tissues from acetate, but the main production sites are the liver, the skin and the intestinal mucosa. Steroid hormone formation in endocrine glands probably relies mostly on exogenous cholesterol (plasma cholesterol). The 27-carbon skeleton of cholesterol is derived from acetyl-CoA through a series of reactions which involve the following intermediate products: (1) Mevalonate (by condensation of 3 molecules of acetyl-CoA), which requires the enzyme HMG-CoA-reductase, an important enzyme in the control of cholesterol biosynthesis; (2) Squalene, a 30-carbon linear structure which undergoes cyclization to yield (3) Lanosterol; and (4) after removal of 3 carbons, cholesterol.

From cholesterol to progestins, androgens and estrogens

The first committed step in steroid biosynthesis is the conversion of the 27-carbon skeleton of cholesterol to a C21-compound, pregnenolone (Fig. 2). This critical step, which is subject to hormonal control by adrenocorticotropic hormone (ACTH) in the adrenals and by luteinizing hormone (LH) in the gonads, is catalyzed by a P-450 enzyme, the cholesterol side-chain cleavage enzyme P-450scc (also called 20,22-desmolase, or 20,22-lyase). Pregnenolone can be converted either to progesterone, which branches to the glucocorticoid and androgen/estrogen pathways, or to 17�-hydroxypregnenolone, which is another route for the formation of androgens and estrogens (Fig. 2, top-left part). Androgen formation in the adrenals is limited to dehydroepiandrosterone and androstenedione, whereas in the testes 17ß-hydroxysteroid dehydrogenase (17HSD) in Leydig cells (under the control of LH) stimulates production of testosterone, the principal "male" hormone. Estrogen formation requires another P-450 enzyme, the aromatase complex (P-450Arom). The substrate is either androstenedione (for estrone) or testosterone (for estradiol). Estrone and estradiol are interconvertible through a reversible reaction involving another 17ß-hydroxysteroid dehydrogenase, as in the androstenedione-testosterone conversion. Aromatase activity is present in the ovary and the placenta. In the ovary, aromatase activity and estrogen formation occur in granulosa cells and are controlled by follicle-stimulating hormone (FSH), whereas production of the androgenic substrates (testosterone, 4-androstenedione) requires LH stimulation of the theca cells.

From progesterone and 17-hydroxyprogesterone to gluco- and mineralocorticoids.

Hydroxylation of progesterone at carbon 21 yields 11-deoxycorticosterone (DOC), and corticosterone after another hydroxylation step at carbon 11. Corticosterone is a major glucocorticoid in rats and other species (e.g., birds), which do not produce cortisol. Two further steps (hydroxylation and oxydoreduction at carbon 18) result in the formation of aldosterone. Cortisol is formed from 17�-hydroxyprogesterone, with 11-deoxycortisol as an intermediate. Cortisol is the main glucocorticoid secreted by the adrenal glands in most mammals.

Steroid hormones in the blood

It is generally assumed that steroids are released into the blood circulation as soon as they are formed, i.e. there are no active transport and/or release mechanisms. Secretion rates are therefore directly related to the biosynthetic activity of the gland and to the blood flow rate.

Steroid binding proteins

Because of their lipophilic properties, free steroid molecules are not highly soluble in water. In biological fluids they are usually found either in a conjugated form, i.e. linked to a hydrophilic moiety (e.g. as sulfate or glucuronide derivatives) or bound to carrier proteins (non-covalent, reversible binding). Binding to plasma albumin (which accounts for 20-50% of the bound fraction) is rather unspecific, whereas binding to either corticosteroid-binding globulin (CBG) or sex hormone-binding globulin (SHBG) [sometimes called "sex steroid-binding protein", or SBP] is based on more stringent stereospecific criteria. The "free fraction" (1-10% of total plasma concentration) is usually considered to represent the biologically active fraction (i.e., hormone that is directly available for action), although this idea has been challenged by recent evidence that, in some cases at least, the specific binding proteins may facilitate steroid entry into target tissues. Apart from the two functions mentioned above, the major roles of plasma binding proteins seem to be (a) to act as a "buffer" or reservoir for active hormones (because of the non-covalent nature of the binding, protein-bound steroids are released into the plasma in free form as soon as the free concentration drops according to the


law of mass action) and (b) to protect the hormone from peripheral metabolism (notably by liver enzymes) and increase the half-life of biologically active forms.

Peripheral metabolism of circulating steroids

Because steroids are lipophilic, they diffuse easily through the cell membranes, and therefore have a very large distribution volume. In their target tissues, steroids are concentrated by an uptake mechanism, which relies on their binding to intracellular proteins (or "receptors", see below). High concentrations of steroids are also found in adipose tissue, although this is not a target for hormone action. In the human male, adipose tissue contains aromatase activity, and seems to be the main source of androgen-derived estrogens found in the circulation. But most of the peripheral metabolism occurs in the liver and to some extent in the kidneys, which are the major sites of hormone inactivation and elimination, or catabolism (see below).

Steroid interaction with target tissues Formation of active metabolites in target tissues

For certain classes of hormones and particular target tissues, steroids must be converted in situ to an active form before they can interact with their specific receptor(s). For example, conversion of testosterone to 5�-DHT (Fig. 3, top) is required for its action on prostate growth and function, whereas aromatization to estradiol-17ß in the brain is mandatory for some of its developmental, neuroendocrine and behavioral effects. Unlike its parent compound, the progesterone metabolite 5�-DHP (Fig. 3, bottom) has no effect on the uterus, but is more effective than progesterone itself regarding the facilitation and/or inhibition of GnRH-induced LH release in vitro. The two main classes of hormones for which metabolic activation has been shown to play a role are the progestins and the androgens, but catecholestrogens (2- or 4-OH derivatives of estrogens) may also constitute another class of biologically active compounds resulting from target organ metabolism.

Correlation between structure and function: the role of metabolism

The biological activity of a steroid molecule depends on its ability to interact with a specific binding site on the corresponding receptor. In most cases, biological activity can be directly correlated with binding affinity. The affinity (usually characterized by the binding constant KD, which is the molar concentration required to saturate half of the available binding sites) of a steroid for its specific receptor is dependent upon the presence or absence of particular functional groups and the overall three-dimensional structure of the molecule. Stereoisomerism may play an important role in this respect: molecules with the same chemical composition but a different spatial orientation of their substituents may have totally different binding properties and biological effects. Thus, 5�-reduced dihydrotestosterone (DHT) is a potent androgen, with a strong affinity for intracellular androgen receptors, whereas its 5ß-epimers do not bind to these receptors and are totally devoid of androgenic properties.

Steroid inactivation and catabolism General principles

Inactivation refers to the metabolic conversion of a biologically active compound into an inactive one. Inactivation can occur at various stages of hormone action. Peripheral inactivation (e.g., by liver enzymes) is required to ensure steady-state levels of plasma hormones as steroids are more or less continuously secreted into the bloodstream. Moreover, if a hormone is to act as a "chemical signal", its half-life in the circulation must be limited so that any change in secretion rate is immediately reflected by a change in its plasma concentration (particularly when secretion rates are decreased). But hormone inactivation can also occur in target tissues, notably after the hormone has triggered the relevant biological effects in order to ensure termination of hormone action. The main site of peripheral steroid inactivation and catabolism is the liver, but some catabolic activity also occurs in the kidneys. Inactive hormones are mainly eliminated as urinary (mostly conjugated) metabolites. Usually, steroids are eliminated once they have been inactivated (i.e., they are not "recycled"). This elimination requires conversion to hydrophilic compounds in order to ensure their solubility in biological fluids at rather high concentrations. A few examples of steroid excretion products are shown in Table 1.

Formation of steroid conjugates

Conjugation (formation of hydrophilic molecules) is an important step in steroid catabolism. Most excretory products in urine are in conjugated form. Two major pathways are used:


1. Formation of glucuronides. This reaction requires uridine diphosphoglucuronic acid (UDPGA) and a glucuronyl transferase. Glucuronic acid is attached to a HO-group on the steroid molecule.

2. Formation of sulphates. This conversion is catalyzed by sulphokinases, which occur in the cytosol of liver, testicular, adrenal and fetal tissues. The substrates are steroids with an HO-group and phosphoadenosine-5’-phosphosulphate (PAPS).

Two examples of conjugated derivatives are shown in Fig. 4. In addition to being excretory products, sulphates are also found in endocrine tissues and/or the plasma as precursors for hormone synthesis. This is the case of dehydroepiandrosterone sulphate (DHEAS), which is used notably for estrogen biosynthesis in the fetoplacental unit. Sulphatases occurring in the microsomal fraction of liver, testis, ovary, adrenal and placenta catalyze the hydrolysis of sulphated steroids to free steroids. The digestive juice of the snail Helix pomatia contains both sulphatase and glucuronidase activity, and extracts from this source are used to hydrolyse urinary conjugates in vitro for clinical assessment of total and conjugated excretion products.

In most species, fecal steroid metabolites are not excreted in a conjugated form, although exceptions exist (e.g., felids).

Summary

Metabolism plays many important roles in steroid hormone action. Various biosynthetic pathways occurring in endocrine glands such as the gonads, the adrenals and the fetoplacental unit are required to produce and secrete circulating hormones. These hormones are partly metabolized in the periphery, either before reaching their target tissues (to control plasma levels of active compounds), or after termination of their action (inactivation and elimination). But many of them are also metabolized within their target tissues, where a complex interplay between activation and inactivation mechanisms serves to regulate the specificity and the amplitude of the hormonal response. The proportion of steroid excreted in urine or feces usually is species or taxon specific. For example, most felids excrete >90% of gonadal steroids into feces, whereas baboons excrete >80% of gonadal steroids into urine. Steroids vary in the extent to which they are metabolized before excretion, both within and among individuals and species. The time course of steroid excretion and the degree to which steroids are excreted in urine or feces are determined by infusing unlabeled or radiolabeled steroid and quantifying hormonal metabolites in excreta. The lag-time from steroid production/secretion to appearance in excreted urine is generally <12 hours, but can range from 12-24 hours in ruminants and 24-48 hours in primates and hindgut fermenters.


1a 1b

FIGURE 1. Structure and classification of steroid hormones. (a) Top: structure of cholesterol, with numbering of carbons (1 to 27) and identification of cycles (A to D). Bottom: The three main classes of steroids ("parents compounds") derived from cholesterol, with number of carbons in brackets (C21 to C18). Pregnanes (C21) form the basic structure of progestins and corticosteroids, Androstanes (C19) of androgens and Estranes (C18) of estrogens (with of phenolic A- ring). (b) Three-dimensional conformation of naturally occurring steroid hormones. Functional groups (or hydrogen atoms) are either in axial (a) or equatorial (e) position. Functional groups (or hydrogens) above the plane of the molecule are said to be in the "ß " position (e.g. the HO-group at C), those below the plane in "�" position (e.g. the hydrogen at position C5, bottom figure). Orientation of substituents at certain positions can be critical for the overall conformation of the molecule. Thus, orientation of the hydrogen atom at the A-B ring junction (marked by open circles in Fig. 1b) will determine whether the A and B rings are fused in a trans- (as in the molecule on top) or cis- (bottom drawing) conformation. This type of conformation (trans- or cis- A-B ring fusion) is often critical for biological activity. For example, 5�-DHT (see Fig. 3) has androgenic properties not shared by its 5ß-reduced analog, 5ß-DHT.


FIGURE 2. Major Pathways of Steroid Biosynthesis. The pathways outlined here are common to the adrenals, the gonads and, to some extent, to the fetoplacental unit. The first committed step is the conversion of cholesterol to pregnenolone, catalyzed by the P-450scc enzyme, which is under pituitary hormone control (ACTH or LH depending on the tissue). Cholesterol side-chain removal is blocked specifically by aminoglutethimide, a steroid biosynthesis inhibitor. From pregnenolone, steroid biosynthesis can proceed either through the so-called "delta-5" pathway (17�-hydroxypregnenolone, dehydroepiandrosterone, testosterone), or through the "delta-4" pathway (progesterone onwards). Progesterone is the starting point for mineralocorticoid synthesis, whereas glucocorticoids are derived from its metabolite, 17�-hydroxyprogesterone. Estrogens are formed from androgens (androstenedione and/or testosterone). Most reactions are irreversible (as denoted by a single arrow). Reversible reactions (double arrows) depend on cofactor availability (e.g. the NADP/NADPH ratio). [Abbreviations used here for the various enzymes are listed in the figure].


FIGURE 3. Steroid metabolism in target tissues. Two examples showing pathways of steroid metabolism in target tissues, which results in the formation of biologically active metabolites. Top: 5�-reduction (left) or aromatization (right) of testosterone. Bottom: 5�-reduction (left) or 5ß-reduction (right) of progesterone.


TABLE 1. Steroid excretion products (examples)

Steroid class Excretion product Type of conjugate

Progestins Progesterone Pregnanediol Glucuronide

17�-hydroxyprogesterone

Pregnanetriol Glucuronide

Androgens Testosterone Androsterone Etiocholanolone

Glucuronide and/or Sulphate

Glucocorticoids Cortisol 11ß-hydroxyandrosterone Allotetrahydrocortisone

Glucuronide

FIGURE 4. Glucuronide and sulphate derivatives. Testosterone glucuronide (left) and estrone sulphate (right), two steroid conjugates found in human urine as excretory product for testosterone and estrone, respectively.


XIX. REVIEW OF REPRODUCTIVE PHYSIOLOGY

Reproduction is controlled by the complex interaction of hormones within the hypothalamo-hypophyseal-gonadal (HHG) axis as shown in Figure 1. In brief, gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the release of gonadotropins, LH and FSH, from the anterior pituitary gland. In the female, gonadotropins are responsible for follicular development, ovulation and corpus luteum function. In the male, these same hormones stimulate testicular development, spermatogenesis and testosterone production.

In both males and females, products from the gonads (e.g., estradiol, progesterone, inhibin, testosterone) feedback to the pituitary gland and hypothalamus to regulate secretion of the gonadotropins. Thus, gonadal activity is

tightly regulated by the interaction of intrinsic stimulatory factors and resulting products. Reproduction also is affected by external factors, many of which are seasonally mediated (e.g., nutrition, photoperiod, climate). When studying a new species it is important to collect data for periods exceeding one year to determine the influence of season or other extrinsic factors on reproductive activity.

Female reproduction

Ovarian cycle dynamics are particularly complex and mechanisms of control not completely understood. In general, during mammalian reproductive cycles a species-specific number of follicles is selected to complete differentiation and ovulate. This occurs after regression of the corpus luteum and withdrawal of progesterone. In most species, ovulation will not occur until this ‘progesterone block’ is removed. During the early follicular phase in mammals such as cattle, horses and primates, after the recruitment of a cohort of follicles, one follicle is selected to become dominant and continues to grow, while growth of the subordinate follicles is curtailed. Shortly after selection, concentrations of gonadotropin receptors and steroidogenic enzymes increase in the dominant follicle. The granulosa cells of the selected follicle acquire LH receptors to allow them to increase aromatization in response to LH as well as FSH. The increased P450 aromatase activity within the follicle causes an increase in concentrations of estradiol that eventually elicits the preovulatory LH/FSH surge. Recent evidence also suggests that intrafollicular insulin-like growth factor (IGF) synergizes with FSH to promote follicular growth and aromatization and helps complete dominant follicular selection. After ovulation, LH works to reorganize the collapsed follicle into a corpus luteum and initiates and maintains progesterone production. If the female becomes pregnant, the corpus luteum is maintained and progesterone of luteal and/or placental sources sustains the pregnancy. If conception does not occur, prostaglandin-F2� and/or estrogens of uterine and/or ovarian sources cause luteal regression and the ovarian cycle begins again.

OocyteOocyte

GnRHGnRH

LHLHFSHFSH

TestosteroneTestosteroneProgesterone

& Estradiol

Progesterone

& Estradiol

OvaryFollicle development

OvulationCL development

TestisTubule formationSperm production

Leydig cell function

OocyteOocyte

GnRHGnRH

LHLHFSHFSH

TestosteroneTestosteroneProgesterone

& Estradiol

Progesterone

& Estradiol

OvaryFollicle development

OvulationCL development

TestisTubule formationSperm production

Leydig cell function

Hypothalamus

Figure 1. Example of hormonal interactions within the

HHG axis.


Females of some species are known as "induced ovulators" in that a mating or similar stimulus is required to induce the ovulatory LH surge and cause ovulation of the follicle. In these species, follicles grow and then regress with no luteal phase unless an ovulatory stimulus occurs. In many of these species, a non-pregnant luteal phase or pseudopregnancy results after non-conceptive matings. These luteal phases can be of similar (e.g., ferrets) or shorter

(e.g., felids) duration than a pregnant luteal phase.

In developing assisted reproductive techniques, like artificial insemination (AI) and in vitro fertilization/embryo transfer (IVF/ET), hormonal therapies are used to control reproductive processes. Thus, equine chorionic gonadotropin (eCG or PMSG), which has FSH-like activity, can stimulate follicular development, whereas human chorionic gonadotropin (hCG), which has LH-like activity, is used to induce ovulation (see Figure 1). Development of these technologies requires an understanding of the mechanisms controlling ovarian function and relies on the availability of adequate endocrine information to determine how well exogenous chorionic gonadotropin therapy mimics natural responses.

Male reproduction In the male, LH stimulates the production of testosterone from the interstitial cells, also known as Leydig cells. FSH is important for seminiferous tubule formation and works in conjunction with testosterone to stimulate spermatogenesis. Testosterone further acts to maintain secondary sex characteristics (e.g., lion’s mane, facial hair, increased muscle mass, colored plumage, etc.) and accessory sex gland function in part through local conversion to dihydrotestosterone (DHT), and sexual behavior. Non-invasive hormone monitoring

Obviously, the ability to track gonadal activity is essential for understanding the fundamentals of reproduction. Fecal and urinary steroid metabolite monitoring are now well-established tools for evaluating reproductive processes in diverse mammalian species. Steroids are easily extracted from feces by boiling in 90% aqueous ethanol, whereas urinary steroids often are directly measurable by immunoassay using group-specific antibodies that crossreact with excreted metabolites or metabolite conjugates. With assisted reproductive techniques, like AI and IVF/ET, becoming increasingly important for managing species ex situ, steroid metabolite monitoring has provided an especially useful tool for examining the efficacy of associated hormonal therapies on reproductive responses. Because of its enormous utility and noninvasive nature, excreted hormone metabolite monitoring has become one of the most powerful tools available in wildlife research today.

XX. REVIEW OF ADRENAL PHYSIOLOGY

Although not a reproductive hormone per se, the potential impact of cortisol (i.e., stress) on reproduction cannot be overlooked. Since the early 70’s “stress” has become an increasingly popular and widely applied term and usually conjures up negative images; however, disagreements continue over a clear definition of the term. It has been defined as a general syndrome occurring in response to any stimulus that

Follicle Development

Day 0

Ovulation CL Development

The female ovarian cycle – Spontaneous ovulation

EstradiolEstradiolProgesteroneProgesterone

LHLH

FSHFSH

Follicle Development

Day 0

Ovulation CL Development

The female ovarian cycle – Spontaneous ovulation

EstradiolEstradiolProgesteroneProgesterone

LHLH

FSHFSH

Figure 2. Longitudinal secretory profile of the major reproductive

hormones during a spontaneous ovarian cycle.


threatens or appears to threaten the homeostasis (or the physiological and physical integrity) of an individual. The stimuli are called “stressors” and the syndrome “stress”. It also has been described as the “fight or flight” response. This physiological stress response appears to have evolved as an adaptive mechanism that allows organisms to adjust to and cope with less predictable circumstances in their environment and to respond rapidly to a wide variety of stimuli. Thus, “stress” represents an important part of life and should not be considered inherently bad. Some researchers have recently argued that the term “stress” should only be used for events that are detrimental for an individual. However, it often is difficult to distinguish a “normal” and an adaptive response from ones that lead to negative effects. Although we can clearly identify some of the negative effects, we are not yet able to distinguish positive and negative stressors and associated responses reliably.

The perception of a stressor by an organism activates the hypothalamic-pituitary-adrenal (HPA) axis (see Figure 3). The hypothalamus releases CRF (corticotropin releasing factor), which stimulates the pituitary gland to secrete ACTH (adrenocorticotropin), thus causing the release of corticoids (often termed “stress” hormones) from the adrenal gland. A number of acute events such as mating, fighting, chasing, temperature shock, pain, etc. can evoke the stress response and activate the adrenal cortex. The resulting rapid changes in heart rate, blood pressure, and gastrointestinal activity are all designed to allow the organism to quickly respond to the situation. However, it is when acute stress occurs repeatedly without allowing for coping responses or recovery, and/or when the stress response is chronically activated that stress becomes a problem, called “distress”. An accumulation of biological costs through a series of acute stressors and/or a consistent chronic stressor have been shown to lead to various pathological conditions, such as immune deficiency, reproductive suppression, growth reduction, muscle wasting, gastrointestinal dysfunction and impaired brain function. Thus, although acute stress can have a stimulatory or facilitatory effect on certain aspects of reproduction, chronic stress can lead to an overall inhibition of reproduction. Identifying early symptoms of distress and pinpointing chronic as well as repetitive acute stressors is essential for evaluating animal well-being. However, it also must be recognized that responses to stress can be very individual-specific. Animals born and raised in captivity may react very differently compared to their wild counterparts even when faced with the same stressors. In captivity many stressors have been removed, but the lack of stimulation in and of itself may present

a source of distress due to boredom. Measuring stress and distress is difficult. No single biochemical or behavioral measure can be used

to assess animal well-being or stress. Whereas behavioral observations may frequently provide a first indicator of distress, they can also be misleading. Similarly, measures of reproductive success, growth rate, and general health, although important for examining certain aspects of animal well-being, often are not reliable as early indicators of distress when examined by themselves. Only through a combination of these measure, including physiological analyses of circulating and excreted hormones like corticoids, can we begin to examine the influence of stress and distress on animal well-being.

The recent development of non-invasive monitoring of adrenal steroids has provided a new tool for these investigations. Measuring glucocorticoid concentrations in blood samples has long been used as an indicator of stress in mammals. However, the invasive nature and inherent stress of collecting blood samples

has limited its usefulness for studies on many captive and wild animals. Alternatively, fecal or

urinary corticoid monitoring can be used in combination with behavioral observations and other measures of overall health in longitudinal studies without additional stress to the animal. Careful biochemical and physiological validation is necessary for the application of this technique and resulting measures cannot provide a “litmus” test for distress. Increases in excreted glucocorticoids may be due to “negative” (i.e., nonadaptive) or “positive” (i.e., adaptive) stress responses. Furthermore, glucocorticoid levels may decline due to intrinsic hormone control and negative feed back mechanisms rather than elimination or decrease of the external stressor. In addition, although a wide variety of stressors stimulate the HPA axis, not all types of

Adrenal

Gland

ACTHACTH

CRFCRF

CortisolCortisol“Fight or Flight” response

Immune suppression

Reproductive inhibition

Anterior

Pituitary

Hypothalamus

Adrenal

Gland

ACTHACTH

CRFCRF

CortisolCortisol“Fight or Flight” response

Immune suppression

Reproductive inhibition

Anterior

Pituitary

Hypothalamus

Figure 3. Hormonal interactions

within the HPA axis.


stressors will affect an increase in glucocorticoids. Nevertheless, the combined use of all available measures can help us better understand the impact of various stressors on animal well-being and make significant advances in our assessment of distress.

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