Laboratory Methods in Endocrinology

I. SURGICAL AND CHEMICAL METHODS

1. Ectopic transplantation of a gland (e.g., under kidney capsule, or to an eye)

2. Chemical ablation (e.g., in pancreas use alloxan kills b-cells, cobalt chloride kills a-cells)

3. Surgical ablation (e.g., hypophysectomy (hypophysectomy), gonadectomy

4. Compensatory hypertrophy (e.g., after the removal of one gonad) (Fig.4.1)

(5. Hormone replacement therapy (e.g., estradiol at menopause to reduce osteoporosis.)

II. IMMUNOLOGICAL NEUTRALIZATION

1. Prepare antibodies against peptide hormones (for male contraception???)

III. EXTRACTION, PURIFICATION, AND SYNTHESIS

1. Early diabetics required daily injections of insulin. (immunological neutralization)

2. Infertility patients could use only human sources of gonadotropins.

2. Agonists (agonist) substitute for a hormone (e.g., progestins that stop GnRH secretion).

3. Antagonists interfere with the action of hormones (e.g., Mifepristone [Mifepristone], or RU486, which blocks P₄ receptor).

IV. HISTOLOGICAL, CYTOLOGICAL & IMMUNOHISTOCHEMICAL METHODS

1. Microscopic studies can provide only general details of anatomical structure, organ size, vascularization, and innervation.

2. Microscopy can reveal if an endocrine tissue is hypertrophic (hypertrophy) (by the ER & GB) (usually with hyperplasia [hyperplasia]), or atrophic [atrophy].

3. Microscopy can also tell if the cell/tissue is acidic (if it responds to a basic dye like hematoxylin which reacts with phosphoric and nucleic acids), or basic (if it responds to acidic dyes like eosin).

4. Immunohistochemistry based on specific antibodies to detect spec. hormones. (Fig.4.2)

a. Immunocytochemistry (or, immunohistofluorescence) uses antibodies conjugated to a fluorescent dye to react with peptide/protein hormones in order to locate the distribution of the hormone in glands/tissues.

b. Immunoenzyme histochemistry uses antibodies conjugated to enzyme (such as peroxidase) and allows the antibody/enzyme complex to interact with a microscopic slice of tissue before the tissue is exposed to a substrate that will develop color when hydrolyzed by the enzyme

V. BIOASSAYS (This was the initial method early workers used to measure hormones)

1. The end-point of a bioassay is some measurable change in metabolism of a target tissue.

2. Examples include:

(a) an insulin-induced decrease in blood glucose in fasted rodents

(b) thyroxine-induced metamorphosis in tadpoles

(d) hCG-induced ovulation in a frog or rabbit.

VI. RADIOIMMUNOASSAYS AND COMPETITIVE BINDING ASSAYS

1. To carry out such an assay, one must have access to three principal components:

(a) first, it is essential to have some specific binding compound (e.g. an antibody, or a receptor molecule that naturally reacts with the hormone, or some protein such as a plasma protein that specifically reacts with the hormone) that will bind to the hormone

(b) secondly, one must have a pure form of the hormone. (for std. curve & for antibody)

2. The “competitive” nature of RIAs, radioreceptor assays, or protein binding assays. In essence, in a competitive binding assay, when an extract containing an unknown amount of the hormone is placed in a reaction mixture with the binding agent (i.e., the "binder") and a known amount of the radioactive hormone (i.e., the "tracer") the unknown hormone competes with the tracer for the binder. (RIA competitive nature)

3. Schematic of a typical RIA procedure (Fig.4.3)

VII. NON-RADIOISOTOPIC IMMUNOASSAYS (i.e., immunometric assays) (Fig.4.4)

1. 1^st antibody is attached to a solid surface, such as wall of a well.

2. Next, incubate (“capture”) the hormone with an epitope corresponding to 1^st antibody.

3. 2^nd antibody is chemically attached to a fluorescent, chemiluminescent, or enzymatic label.

4. Add this signal antibody to incubate, and it attaches to a second epitope on the hormone.

5. The chemical “signal” can then be read as a direct reflection of amount of bound hormone.

VIII. NORTHERN ANALYSES QUANTITATE mRNA LEVELS (Fig.northern)

1. Extract total RNA from experimental tissue and “control” tissue.

2. Separate RNA, by size, by electrophoresis on an agarose minigel.

3. Transfer (by blotting) the mRNA from the agarose to a nitrocellulose membrane.

4. Prepare a radio-labeled probe of cDNA representing the hormone.

5. Allow cDNA probe to hybridize to the mRNA membrane, and expose to film.

IX. IN SITU HYBRIDIZATION SHOWS SPATIAL EXPRESSION OF mRNA

1. Prepare a radio-labeled cDNA or cRNA probe, representing hormone of interest.

2. Prepare a microscope slide with a section of the tissue of interest.

3. Allow radio-labeled probe to hybridize to the mRNA in the microscope slide.

(Fig.x) (Fig.y) (Fig.z)

X. AN OVERVIEW OF A RECOMBINANT DNA TECHNIQUE (Fig.4.11)

1. Purify and insert segments of genetic material (e.g., insulin) into bacterial host(s).

2. Promote DNA synthesis in the bacteria, in order to produce much peptide.

3. Place protein products of a- and b-chains in conditions to promote disulfide bonding.

III. CRITERIA FOR QUANTITATIVE MEASUREMENT

1. Specificity is the ability of an assay to detect only one hormone and exclude others.

2. Accuracy is the closeness of the numerical value of the assay to the true amount of the hormone. (One can estimate the accuracy by adding a known amount of the hormone to an extract and then determine the "recovery" during the assay.)

3. Sensitivity is related to the smallest amount of a hormone that can be measured.

4. Reproducibility refers to the closeness of results when samples are assayed at different times. (The reproducibility will depend on skills of the investigator, nature of the assay, the natural variability of the metabolism of the organism studied, etc.)

5. Intra-and inter-assay coefficients of variation:

a. Intra-assay CV =Std. Deviation/mean (same sample is assayed at the same time)

b. Inter-assay CV = Std. Deviation/mean (same sample is assayed on different days)

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