Prostaglandin E₂ Measurement

New Developments

by Kirk M. Maxey, M.D.

Prostaglandin E₂ (PGE₂) is a primary product of arachidonic acid metabolism in many cells. Endogenously formed PGE₂ is rapidly converted into its inactive 13,14-dihydro-15-keto PGE₂ metabolite (see Figure 1). The half-life of PGE₂ in the peripheral circulation is less than 30 seconds, and normal plasma levels are below 10 pg/ml. PGE₂ produced in vitro is not so rapidly metabolized, resulting in the accumulation of PGE₂ in most culture supernatants. For instance, differentiated HL-60 monocytes in culture can produce ng/ml levels of PGE₂ in a few days. No single analytical method will satisfy the many different research programs with an interest in PGE₂ measurement. Some look for very small amounts of PGE₂ in complex biological samples; others in high throughput NSAID screening favor speed and a broad dynamic range. Recent advances in our PGE₂ research assay systems can benefit nearly all of them.

PGE₂ Monoclonal: The Gold Standard

The best all around PGE₂ assay continues to be the Cayman EIA based on the 2B5 monoclonal antibody to PGE₂. This monoclonal anti-PGE₂ antibody binds PGE₂ with the highest affinity ever reported for this substance in the literature. It was the use of this antibody in animal models of inflammation that led to the introduction of a new paradigm: "PGE₂ = inflammation". Recently we showed that by simply lowering the incubation temperature for this assay to 4ºC, it was possible to obtain even better sensitivity. Figure 2 shows the influence of this modification on the standard curve. With an IC₅₀ of 54 pg/ml, this assay is a full log more sensitive than other PGE₂ assays based on less specific antibodies.

PGE₂ Affinity Sorbent

With a detection limit of 10 pg/ml, our PGE₂ monoclonal EIA approaches the concentrations relevant in biological specimens. However, it is well known that matrix effects and interference in samples such as plasma and serum require dilutions of 10 to 50-fold before PGE₂ can be measured. Now a powerful new purification technique can bring these concentrations back into the standard curve range while making sample cleanup quick and easy.

Cayman has covalently attached the monoclonal anti-PGE₂ antibody to Sepharose 4B, creating a versatile, high affinity reagent. PGE₂ Affinity Sorbent is unique in its ability to isolate and purify trace amounts of PGE₂ from complex samples. In a typical experiment, 1 ml of PGE₂ Affinity Sorbent is stirred for 30 minutes with a 10 ml plasma sample containing from 0.1 - 1000 pg/ml PGE₂. More than 99% of the PGE₂ in the sample will be retained on the beads. This bound PGE₂ can be easily recovered by centrifugation followed by an aqueous alcohol wash. PGE₂ Affinity Sorbent can be used to strip PGE₂-immunoreactivity from samples. It can also be used in a conventional column format if desired. PGE₂ Affinity Sorbent makes the analysis of complex samples for PGE₂ practical for the first time.

NSAID High Throughput Screening

As already mentioned, searching for inhibitors of cyclooxygenase has generated a large market for PGE₂ assay methods. The needs and constraints of this market segment are fundamentally different from any other. Using mitogens to stimulate the induction of COX activity and frequently using cells transfected with the human COX-2 enzyme, these researchers generate thousands of samples with ng/ml PGE₂ concentrations. Other prostaglandins such as 6-keto PGF_1α and PGF_2α may also be produced.

Using a synthetic hapten analog produced by the Cayman organic chemistry division, we recently developed a unique antiserum with a broad range of affinity for prostaglandins. Our new PG Screen EIA is based on this polyclonal antiserum. The assay features a dynamic range of nearly 4 logs, from a detection limit of 24 pg/ml to an upper limit above 10 ng/ml (See Figure 3). The assay detects PGF_2α, PGE₂, and PGE₁ with equal affinity. It also crossreacts significantly with PGD₂ and 6-keto PGF_1α, making it a powerful tool for the measurement of total prostaglandin production. The assay is available in both colorimetric and chemiluminescent formats.

Cayman to Aid University with Sea Lamprey Research

by Kirk M. Maxey, M.D.

The invasion of the sea lamprey into the Great Lakes through the Welland canal at the turn of the century is commonly recognized as the greatest aquatic ecological disaster engendered by mankind. This primitive, predatory fish native to the Atlantic thrived on the abundant populations of Great Lakes trout and whitefish, which were also the basis for an important commercial fishery employing thousands. By the 1940s, fish populations were devastated and the fishery collapsed. Intensive government efforts to eradicate the sea lamprey had some success, but were never able to effectively remove the lampreys or restore the commercial fishery. Direct expenditures by the American and Canadian governments for lamprey control now total nearly $12 million each year. An international agency, the Great Lakes Fisheries Commission, now funds and oversees this effort. The mainstay of present lamprey control is chemical poisoning of the streams where sea lampreys spawn, using the lampricide trifluoromethyl nitrophenol. An experimental program to further control lamprey populations through the capture and release of 25,000 sterilized males may also be helping, but the problem is far from solved. Lamprey populations in the lakes are stable and have declined little in recent years. A number of river systems, including the Saint Mary's River, are acknowledged by fisheries officials as being infested with uncontrolled populations of lampreys.

The sea lamprey (Petromyzon marinus) belongs to a very primitive group of jawless fish known as the agnatha. They lack both a jaw and a backbone, and their complex life cycle includes a worm-like larval stage. Sea lamprey larvae bury themselves in the mud on the bottom of the streams where they hatch, and can remain there, feeding and maturing slowly, for up to fifteen years. They then undergo a radical metamorphosis which transforms them into an eel-like, parasitic juvenile animal with a hook-studded sucker for a mouth and a strong appetite for trout and salmon.

Recently, researchers at the University of Minnesota have discovered an aspect of the lamprey reproductive cycle which may permit the development of new and safer control measures. Like the chemical lampricides, which target the larval stage of the lamprey in order to avoid killing desired fish, the new findings also focus on this unique part of the lamprey life cycle. In a migration somewhat reminiscent of the well known salmon runs, the sea lamprey returns to spawn in the shallow streams which have supported spawning in the past. Researchers now believe that the salmon achieve their miracle of migration by imprinting on a "bouquet" of unique odors and trace chemicals in their native stream shortly after hatching. As adults, they remember this bouquet and can home in on it from hundreds of miles away. In contrast, adult lamprey appear to instinctively recognize the odor of juvenile lamprey in streams, and use this as a cue. Thus, they do not necessarily return to their native stream.

The key to adult lamprey orientation appears to be an exquisite ability to smell the excreted bile acids of their offspring. The larvae have a complete digestive system, including a gall bladder, through which they release a number of sulfated sterols into the water. These chemicals are not only unique to the sea lamprey, but are also unique to the larval stage, since the gall bladder is discarded in the metamorphic transition to a parasitic juvenile that feeds on the body fluids of other fish. Experiments conducted by Dr. Peter Sorensen and his colleagues have shown that one particular compound, known as petromyzonal sulfate (PZS), appears to be a spawning chemoattractant for the sea lamprey. Rather than homing in on the natural "bouquet" of their spawning stream, sea lampreys apparently just home in on the smell of PZS released by their offspring.

Scientists at Cayman Chemical have recently teamed up with Dr. Sorensen's lab in an effort to exploit this discovery for better sea lamprey control. Cayman has undertaken to develop a sensitive immunoassay method for measuring PZS in stream water with the same threshold of detection displayed by the lamprey adults. Members of the Sorensen team at the Department of Fisheries and Wildlife lab in Minnesota had to dissect the gall bladders of over 1,000 larval sea lampreys in order to obtain enough PZS for the assay development. Cayman is using some of this precious natural substance to immunize rabbits. The goal is to produce an antibody which binds specifically to PZS, which is a key part of the assay. Jim MacDonald, who heads the Cayman assay development group, expects to have a working prototype of the assay kit sometime this summer. Cayman chemists will also be analyzing the remaining natural PZS from the larvae in order to develop synthetic chemical reactions which will allow both the completion of the assay, and eventually the laboratory synthesis of PZS.

There are several ways that these discoveries can be put to use for better lamprey control. First, the PZS assay can be used as a quick, accurate, and relatively inexpensive check on the populations of juvenile lamprey larvae in tributary streams. This census must now be done through labor-intensive and error-prone manual catching and counting of the larvae. Accurate population estimates are essential in order to apply the toxic chemical lampricides effectively. Not having an accurate measurement of larval density means that some streams infested with larvae may be skipped because the census takers did not find them. The assay will also be extremely useful for research purposes. It could be used to predict those streams expected to see heavy adult lamprey spawning runs, based on the levels of PZS in the water.

A successful synthesis of PZS by the Cayman organic chemistry team could also open the possibility for sea lamprey traps, using the spawning chemoattractant as bait. Since the lampreys do not appear to really recognize their true birth place, but home in on this single chemical attractant instead, they could be fooled. Blind ditches ending in netted traps could be constructed in stream banks and baited with PZS during the fall, when the adults are spawning. Since sport fish cannot detect PZS, these traps might prove very effective in eliminating only the lamprey adults.

The work of Dr. Sorensen and his colleagues has been funded by the Great Lakes Fisheries Commission in Ann Arbor and Sea Grant. For more information, you can visit their website at http://www.glfc.org. More information is also available from the Cayman Chemical website.

Additional Figures