Assignment #4 - Review of a paper on Parathyroid Hormone

Paper:

Kurland, E.S., Cosman, F., McMahon, D.J., Rosen, C.J., Lindsay, R., Bilezikian, J.P. (2000). Parathyroid Hormone as a Therapy for Idiopathic Osteoporosis in Men: Effects on Bone Mineral Density and Bone Markers. The Journal of Clinical Endocrinology & Metabolism, 85(9): 3069-3078.
Summary:

Osteoporosis is a common and well-known condition primarily affecting post-menopausal women. There are, however, a significant proportion of cases in which osteoporosis affects middle-aged men, consuming more than 2 billion dollars in health care costs and accounting for 20% of fractures in the U.S. annually. Of all male cases, 40% are determined to be idiopathic; that is, with unknown determinate cause.

It is important that male cases and female cases be approached differently because the disease causing mechanisms are speculated to be quite different. Female osteoporosis has symptoms caused by factors that increase bone turnover, whereas idiopathic male osteoporosis involves depressed bone turnover, though the mechanisms causing this decrease are yet to be determined. Although antiresorptive medications, like estrogen and calcitonin, may be effective in female forms of the condition, they are proving to be inefficient or unsuitable to treat affected males. The nature of a low bone-turnover pathological is state requires an anabolic agent to restore or rebuild lost materials.

This study was conducted over an 18 month period with 23 male subjects diagnosed with idiopathic osteoporosis. Their ages ranged from 30 to 68 with a mean age of 50. All patients recieved 1500 mg of calcium and 400 units of vitamin D each day, and ten randomly chosen subjects of this group recieved 400 units of PTH in addition to this regimen. Every 3 months serum, urine and bone markers for turnover were analysed, while every 6 months bone densities were measured at three points commonly affected by osteoporosis in men; hip, lumbar spine and radius (forearm).

Parathyroid hormone is known to stimulate bone resorption when serum calcium is low by indirectly stimulating osteoclast bone cells to resorb bone. PTH acts on the osteoblast, or bone forming cells, which then stimulate osteoclasts to proliferate. While it may seem counterintuitive that treating osteoporosis patients with PTH would increase bone density, administering PTH was found to significantly reverse the effects of bone loss in the hip and lumbar spine in male patients as compared to control subjects. After 18 months, lumbar spine bone mass increased by 13.5% (p<0.001),>

The results observed in this study along with other studies have led to the conclusion that PTH stimulates skeletal dynamics in males with osteoporosis associated with low bone turn-over, by increasing bone mass in hip and spine. The experimenters thus conclude that PTH has potential as an anabolic agent to treat male idiopathic osteoporosis.

Critique

- The paper was well written and related the value of discovering anabolic treatments for male forms of osteoporosis as they are often overlooked and underestimated in their prevalence.

- The experimental design was excellent in terms of reducing error/bias. Subjects were placed into groups randomly, and within these groups well matched on clinical characteristics such as age, BMI, smoking, exercise and previous drug history. The subjects to recieve PTH treatment were chosen randomly from the groups. The study was double-blind and placebo-controlled.

- The researchers cited a previous study that had measured similar features of this PTH regimen and obtained success, however there were considerable flaws in the experimental design. This study was done as a follow-up with some variations in an attempt to fix the experiment to determine if their results were significant.

- In terms of changing the initial protocol, 2 of the 10 patients treated with PTH were found to have elevated serum calcium during the course of the experiment. The dosage for these patients was diluted to normalize calcium levels, yet they do not explain how this change affected the measurement of serum calcium levels that they found not to be significant during treatment. They cited the decreased dose while noting that even by lowering the amount of PTH given, bone density still rose for these subjects.

Possible Future Experiments:

Question: What is the effect on other bones of the body that could be affected by osteoporosis in male subjects?

- It would be useful to undertake more extensive studies of other bones, typically those under postural stress like the femur, to determine if there would be increased bone mass or no change like that of the radius in the study. It would be a huge setback if the treatment were to be marketed and it was found that some bones had increased densities while other had losses.

Question: Though the treatment worked for the three bones studied, did the overall bone mass measured for the individuals rise significantly?

- This could easily be measured as there are instruments available as compact as special scales to measure bone mass of the entire body. However, it is unknown if the bone density increases would have been significant when the entire body was taken into consideration.

Assignment #3 - Function and Pathology of Parathyroid Hormone

Function of Parathyroid Hormone

Parathyroid hormone functions to maintain calcium homeostasis by elevating concentrations through a variety of mechanisms when calcium-sensing receptors detect a drop in serum levels (Griffin & Ojeda, 2004). PTH also functions to decrease concentrations of phosphate ions by reducing phosphate reabsorption in kidney tubules; thus levels are lowered when phosphate is lost in urine (Hadley & Levine, 2006).

In order to raise serum Ca2+ and lower PO4-3, parathyroid hormone acts either directly or indirectly through mechanisms that incorporate bone, kidney and intestines (Hadley & Levine, 2006). These actions can be categorized as follows:


PTH on bone

PTH plays a role in mineral metabolism in order to raise serum calcium should it become low. As PTH is known to release calcium from bone into plasma, at first it seems counterintuitive that stromal osteoblast cells of the bone marrow are stimulated in the presence of the hormone. However, in response to PTH the osteoblast cells will increase expression of RANKL, a molecule that will bind to the RANK receptor on osteoclast precursor molecules, causing them to differentiate into mature cells. The creation of new osteoclasts increases bone resorption, a process that releases free Ca2+ to the serum (Hadley & Levine, 2006). This breakdown of bone, or osteolysis, also releases phosphate into circulation, but at lesser amounts than that of phosphate leaving the body through excretion.





PTH on renal tubules of the kidney

Parathyroid hormone also increases reabsorption of calcium in the proximal tubule of the kidney (Hadley & Levine, 2006).

PTH on intestine, indirectly through kidney

PTH stimulates the kidney to activate vitamin D by up-regulating the enzyme responsible for converting 25-hydroxy vitamin D, to its active form (1,25-dihydroxy vitamin D) in a hydroxylation reaction (Griffin & Ojeda, 2004). The active form of vitamin D is then able to absorb more calcium in the intestine.




Pathology

Hyperparathyroidism:

Overactivity of one or more parathyroid glands leading to excessive PTH secretion (Griffin & Ojeda, 2004). The excess PTH causes a marked increase in bone resorption and calcium absorption in the intestine leading to hypercalcemia in the blood as well as low phosphate levels.
Symptoms include those connected to high plasma calcium levels, for example, renal stones as a result of excessive calcium in the kidney and neurological symptoms since calcium plays such a large role in the nervous system. Weak bones and osteoporosis-like state are also associated with hyperparathyroidism due to a net resorption of minerals from the bones.


Causes include adenomas or PTH-secreting tumors, classified as primary hyperparathyroidism, overactive osteoclasts or a significantly higher production of active vitamin D than normal. In this case, the overactive gland can be surgically removed or calcitonin supplements given to maintain calcium homeostasis.

Hyperparathyroidism effects

Hypoparathyroidism:

Decreased activity of parathyroid glands resulting in low PTH levels and hypocalcemia (Griffin & Ojeda, 2004).

Low serum calcium can be dangerous because most cells, including neurons, require calcium to exert any effect on bodily functions. Thus, many neurological and neuromuscular symptoms are seen, such as seizures, nerve and skin pain, tetany of muscles and muscles spasms, including those muscles in the pharynx.

Hypoparathyroidism can result due to the accidental removal of one or more parathyroid glands during surgery (i.e. during a thyroidectomy). Autoimmune disorders may result in destruction of the parathyroid glands as well as affecting other organs. DiGeorge syndrome is a genetic disorder resulting in deletion of the locus coding parathyroid glands. A severe vitamin D deficiency can also result in hypoparathyroidism, and is reversible by increasing vitamin D consumption. Idiopathic forms also exist and are caused by an autosomal recessive inheritance pattern. In addition, pseudohypoparathyroidism results in the same lowered blood calcium, but it is due to the target organs upon which PTH acts being resistant to the hormone. Diet modifications are important in treatment, and intravenous calcium may be given with caution.

References:

Griffin, J. E., Ojeda, S. R. 2004. Textbook of Endocrine Physiology. Oxford University Press, New York.

Hadley, M. E., Levine, J. E. 2006. Endocrinology, 6th Ed. Pearson Prentice Hall, New Jersey.

Assignment #2: Structure of PTH

Structure of PTH

Parathyroid hormone is encoded by a gene on the short arm of chromosome 11 in humans (Many, 2005). The gene for the other calcium homeostasis hormone, calcitonin, is also located on the same arm of this chromosome, however it is not known if this is significant to regulation.

PTH is an 84-amino acid polypeptide derived from a prohormone (Griffin & Ojeda, 2004). The initial preProPTH is a 115-amino acid chain that is cleaved to ProPTH and then the remaining 6 amino acids at the N terminal are cleaved to produce PTH (Many, 2005). It has been shown in various experiments that the conversion of PreProPTH to PTH occurs in the Golgi apparatus of parathyroid gland chief cells and the reaction is catalyzed by a trypsin-like protease (Hadley & Levine, 2006). The final polypeptide, with a molecular weight of 9500 Da, is packaged in a secretory vesicle migrates to the cell periphery when the gland is stimulated. The hormone is released from the cell by exocytosis.




The degree of similarity in the amino-acid sequence of a hormone (or precursor in this case) found across various genera is a valuable tool to assess differences and importance of the hormone’s function. This type of comparison can also be used to assess evolutionary conservation and infer phylogenetic relationship based on the degree of similarity, as seen in Figure 2 below. Blast and ClustalW were used to obtain protein sequence alignments for the PreProParathyroid hormone between cat, pig, horse, human and mouse genera, as shown in Figure 1.

Key:
* Amino acid residues that are identical between all species compared
: Substitutions which are conserved
. Substitutions which are semi-conserved

Color Key:
RED (AVFPMILW) - Small (small+ hydrophobic (including aromatic -Y))
BLUE (DE) - Acidic
MAGENTA (RK) - Basic
GREEN (STYHCNGQ) - Hydroxyl + Amine + Basic – Q

Figure 1. CLUSTAL 2.0.8 multiple sequence alignment for PreProParathyroid hormone in cat, pig, horse, human and mouse genera.



Figure 2. Phylogenetic comparison of Homo, Mus, Eqqus, Canis and Sus based upon the degree of similarity in the Parathyroid hormone precursor

The PTH receptor has a seven-transmembrane helical structure on which both PTH and another protein, PTHrP (PTH related peptide) bind and induce conformational changes (Many, 2005). This receptor is coupled to G-proteins and stimulates adenylyl cyclise and phospholipase C in presence of bound PTH. There is evidence that the N-terminal region of PTH is critical in order to activate the receptor, and amino-acid residues 17 to 31 are needed for the peptide to bind to the receptor with high affinity.

References:

Griffin, J. E., Ojeda, S. R. 2004. Textbook of Endocrine Physiology. Oxford University Press, New York.

Hadley, M. E., Levine, J. E. 2006. Endocrinology, 6th Ed. Pearson Prentice Hall, New Jersey.

Many, T. N. 2005. Molecular Biology of the Parathyroid. Kluwer Academic/Plenum Publishers, New York.

Assignment 1: Description of PTH

Parathyroid Hormone

Parathyroid hormone, in association with calcitonin and vitamin D, is responsible for regulating calcium metabolism in the body (Griffin & Ojeda, 2004). In adults, the primary action of PTH is to raise serum calcium levels when the body senses a low concentration of ionized calcium in the blood. To induce a response that raises extracellular calcium levels, PTH acts on bone, kidneys and intestine (Many, 2005) in the following ways:

(i) Stimulating specific bone cells that release stored calcium
(ii) Increasing calcium absorption from the gut in a pathway involving vitamin D (1,25-(OH)2 D)
(iii) Acting on the kidney to enhance the calcium being reabsorbed from the urine (Hadley & Levine, 2006).

The parathyroid glands, which secrete parathyroid hormone,

are small pea-sized glands found posterior to the thyroid gland (Griffin & Ojeda, 2004). Four of these glands are normally found, but a small percentage of the population can have as few as two or as many as eight. The parathyroid gland produces PTH by first synthesizing pre-pro-PTH which is cleaved to pro-PTH and then to PTH, an 84-amino acid polypeptide. The hormone is secreted to the bloodstream directly after it is produced and has a half-life of less than 5 minutes in the serum.

The amount of PTH produced depends on the ionized calcium concentration of the blood and is regulated by a negative feedback mechanism (Griffin & Ojeda, 2004). In another feedback loop, the amount of vitamin D directly inhibits PTH secretion. These feedback loops are important to protect the individual from hypercalcemia. Due to these processes, the calcium concentration of blood is kept within a narrow range optimal for proper functioning (Hadley & Levine, 2006).

References:

Griffin, J. E., Ojeda, S. R. 2004. Textbook of Endocrine Physiology. Oxford University Press, New York.

Hadley, M. E., Levine, J. E. 2006. Endocrinology, 6th Ed. Pearson Prentice Hall, New Jersey.

Many, T. N. 2005. Molecular Biology of the Parathyroid. Kluwer Academic/Plenum Publishers, New York.

Introduction

My name is Meghan O'Leary and I'm a fourth year biology major at Memorial University in St. John's, Newfoundland, currently studying and working towards a B.Sc. (honours). This blog has been created as an assignment for my Biology 4605 (Endocrinology) course and will be updated periodically during the semester.

I have chosen to investigate the function, structure and recently published literature on human parathyroid hormone (PTH) as well as the pathology of disorders associated with this hormone.

Below is a picture of bone tissue as a clue to some processes that PTH is involved in.

More information on parathyroid hormone to come!

Meghan