A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro

Authors:

A Sabokbar, P J Millett, B Myer, N Rushton

Abstract:

Studies on osteoblast-like cells isolated from trabecular bone, embryonic calvaria and osteosarcoma have helped to establish a set of properties associated with the osteoblast phenotype. These include alkaline phosphatase activity, synthesis of type I collagen, secretion of osteocalcin and production of a mineralized matrix. Moreover, it has been shown that these osteoblastic characteristics are modulated by dihydroxy vitamin D3, parathyroid hormone, glucocorticoids, prostanoids and various cytokine growth factors.

Of all the above, the most widely recognized biochemical marker of the osteoblast phenotype is alkaline phosphatase (ALP). ALP is a ubiquitous enzyme which catalyses the hydrolysis of phosphate esters at an alkaline pH. In humans there are three isoenzymes: tissue non-specific, intestinal and placental. The tissue non- specific isoenzyme has three isoforms, namely bone, liver and kidney, which contain different carbohydrate moieties on the same polypeptide backbone. Of these iso- forms, bone ALP shares a common protein structure with liver ALP, however, the two can be differentiated by electrophoretic mobility, chemical inactivation, lectin reactivity and thermostability. These differences have been used to distinguish bone ALP from liver ALP in serum and plasma of patients with pathological bone disorders such as osteoporosis or Paget’s disease, while others have used monoclonal antibodies that react preferentially with the bone isoform.

There has been extensive work and publication on ALP over the years, yet the precise function of skeletal ALP in vivo is largely unknown. Evidence suggests that the enzyme is involved in skeletal mineralisation. For instance, ALP has been shown to be associated with the plasma membrane of osteoblasts, which bud out to form the matrix vesicles seen in developing bone. In vitro studies have shown that ALP activity in foetal rat calvaria is proportional to the rate of collagen production. Most conclusive evidence comes from patients suffering from hypophosphatasia, who have a defect in ALP and are characterized by abnormal skeletal mineralization, and from studies in which the introduction of skeletal ALP cDNA into ALP-negative cells conferred an in vitro capacity for mineralization. As yet the precise role of ALP remains largely unknown.

For the complete study: A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro

Peter Millett, MD

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Alexandra Thompson

Mar 23, 2024

by Alexandra Thompson on Peter Millett, MD

110%

Dr. Millett and his team did a SLAP tear repair and bicep tenodesis on my left shoulder. I use my arms as an artist, and love to swim in the ocean, surf, paddle, and telemark ski. So regaining full use of my shoulder and keeping my bicep attached was very important to me. Now, over two years later, my shoulder has the equivalent range of motion as my other arm and as before the injury, and no pain. After the first three months it felt so good and was so strong that - to my horror - I completely forgot my six-month follow-up.

Not only did Dr. Millett and his team make my arm as good as new, but having had the experience of such kindness and expertise throughout the process of surgery and recovery is like an added shoulder superpower.

Thank you Rita, Brooke, Jason, Sarah, Camille, Olivia, Dr. Millett and everyone! And see you soon when my husband comes in for his knee...

Jeff Chandler

Jan 31, 2022

by Jeff Chandler on Peter Millett, MD

Flawless procedure, Easy recovery

Dr. Millet's repaired my knee and tibia, following a botched operation by a different surgeon. That procedure was virtually pain free and I was weight bearing within a few days. That was 18 years ago and the knee is still strong. A few years after the knee incident, Dr Millet repaired my shoulder. My insurance company approved me for 3 months of disability following that surgery. I was off pain medication in 2 days and had 90% range of motion within a week. I withdrew my disability claim and returned to work within two weeks of surgery. I’m not a famous athlete—just a regular guy. Nonetheless, I was treated like an NFL at the Steadman clinic. Most notable was the time for recovery. Other surgeons perform their hour-long task in the OR and don’t involve themselves in the other functions. Peter and his team manages the entire process from diagnosis, anesthesia, nursing care, and therapy. He takes a holistic approach to patient care and doesn’t skip any steps.

Jenn West

Jul 2, 2021

by Jenn West on Peter Millett, MD

Saved My Life

I went to Dr. Millett after 7 knee surgeries (6 on my right and 1 on my left) and had seen 11 different surgeons who all refused to remove femur metal from my right leg. I am highly allergic to metal and the chronic pain was leaving me bedridden for 3.5wks out of a month. He personally wrote me back on my inquiry, listened to my concerns, scheduled my surgery within a week (due to insurance issues) and personally followed up with my recovery. The level of patient care from Dr. Millett and his ENTIRE team is above any care I have ever received in my 20 years of dealing with orthopedic surgeons. There are not enough words to describe the level of care, dedication and professionalism you receive from Dr. Millett and his staff.
My only disappointment is that I did not find him sooner!!!

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A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro

Authors:

Abstract:

About the Author: Peter Millett, MD