Marjorie Groothuis Horning: Biochemist, Pharmacologist, and GC Pioneer

On the occasion of this innovator’s 101st birthday, we look back at her career and her efforts to develop and popularize gas chromatography (GC), particularly in its applications to biomedical research, resulting in early demonstrations of nicotine in the blood of passive smokers, and the transfer of drugs and metabolites from a pregnant mother to the unborn child.

The 100th anniversary communication of U.S. biochemist Marjorie Janice Horning, b. Groothuis, featured in the article "Milestones of Chemistry 2017" in the German magazine Nachrichten aus derChemie, was the occasion to remember her and her contributions to the development of modern biochemical analysis and their importance for raising the awareness of public health issues.

Marjorie Janice Groothuis was born on August 23, 1917, in Detroit, Michigan. She studied at the Goucher College in Baltimore, Maryland (B.A. 1938) and at the University of Michigan in Ann Arbor, where she received her master's degree in 1940, and her doctorate in 1943 with a thesis on the metabolism of phospholipids. As a doctoral student, she met the chemist Evan Charles Horning (1916–1993), who taught at the university. They married in 1942. The Hornings shared a lifelong partnership in scholarly work, as evidenced by over 100 joint publications, as well as common interests in art, travel, and gardening.

After earning her doctorate, Marjorie Horning remained at the University Hospital as a research assistant in pediatrics until 1945. Later on, both Hornings worked at the University of Pennsylvania in Philadelphia for five years, and at the National Heart Institute of the National Institutes of Health (NIH) in Bethesda, Maryland, from 1951 to 1961.

There, the research couple started their pioneering work on modern biochemical analysis. As head of the Natural Products Chemistry Laboratory, Evan recognized that the methods used to date for lipids and steroids were inadequate, and, together with his group, sought more precise and accurate analytical methods for complex biological samples. Particularly promising was the still young method of gas chromatography (GC), because of the combination of separation and detection and the associated possibility of simultaneous analysis of multiple components in one run. The first GC instruments were becoming commercially available and the number of users was rising rapidly.

The method was still in its infancy, however. One worked with packed columns at a constant column temperature. For detection, gas density and thermal conductivity detectors were used; capillary columns, temperature programming, flame ionization detectors, and microliter dosing syringes were introduced in 1958–1959. Positive experiences had been reported mainly for gases and hydrocarbons, but with higher and more polar compounds the results were not encouraging. Long retention times, poor peak shapes, and decomposition on the column created skepticism; some opinion leaders in organic chemistry even doubted that GC might ever be suitable for studying more complex organic compounds.

The Horning group nevertheless achieved breakthroughs by deactivating the silicate support by acid washing and subsequent silanization, using thermally stable, nonpolar stationary phases such as the methyl silicone SE–30. They coated the support with only 1–3% stationary phase, resulting in low film thickness. They achieved passivation of the glass tubes used for column making by silanization, and used glass inserts in the injector or "on-column" injection. With column lengths of 6 or 12 ft, they were able to separate steroids with one to two functional groups within 15–60 min at temperatures of 210–230 °C. Polyfunctional steroids, which were not directly accessible to GC, were converted into less polar, thermostable derivatives by reaction of the polar groups with suitable reagents (hydroxyl to trimethylsilyl ether, keto to methoxime, carboxylic acid to methyl ester), thus making them amenable to GC. All of this work was groundbreaking, both for the development of modern biochemical analytics as well as for extension of the GC application range.

Figure 1: Photo of Marjorie Horning, circa 1987. Reprinted by permission from Springer Customer Service Centre GmbH: Springer Nature (1)

When the Horning's cholesterol work became known to Michael DeBakey, the world famous cardiac surgeon, he invited Evan Horning to Houston, Texas, to lead the newly founded Institute for Lipid Research. Under DeBakey, Baylor College of Medicine would develop into a highly dynamic world-class heart-surgical and medical research center, and also house new institutes with appropriate (bio) chemical–medical alignments developed around the heart center. The presumed importance of cholesterol, mediators such as prostaglandins, and blood lipids or lipoproteins in the formation of atherosclerotic plaques leading to coronary artery occlusion was the scientific link to the main area of medical research at Baylor. Evan Horning insisted that he would only go into the "Deep South" of the United States if Marjorie came with him, and DeBakey agreed. So they moved to Houston in 1961; Evan became director of the institute and professor of chemistry. Marjorie was first an associate professor of biochemistry, and, in 1969, was promoted to professor. She also served as a visiting professor of biochemistry and biophysics at the University of Houston. At Baylor, the elaborated methods were further developed and additional classes of substances were included, such as sugars, sugar alcohols, acids, hormones, and pharmaceuticals and their metabolites. For the latter, so-called profile analyses were developed-that is, parallel and sensitive detection of the largest possible number of metabolites in biological fluids such as blood, urine, or saliva, to distinguish between "normal" profiles and pathological ones (metabolic profiling), and between normal and fast metabolizers. Packed columns were used until 1975, when suitable stable glass capillaries with polar stationary phases became available. The group was also one of the first to use GC–mass spectometry (MS) coupling, initially operated with packed columns. As early as 1966, the Institute was awarded a commercial LKB 9000 instrument, the world's second GC–MS coupling device-a magnetic sector field instrument with electron impact ionization and nozzle separator, according to Becker & Ryhage. In 1969, the first commercial "computer-controlled" GC–MS device, the Finnigan 1015 (quadrupole MS) followed. By means of suitable sample preparation combined with application and further development of chemical ionization, and the so-called "mass fragmentography" (selected ion monitoring [SIM]) and quantification by means of the stable isotope dilution method, they achieved routine measurements in the nanogram to picogram range, starting from a 0.1–1 mL plasma or 1–5 mL urine sample.

The methodological developments in the Horning laboratory were always driven by the intention of applying them in important areas of health care, and when the first mass spectrometer with an atmospheric pressure ionization (API) ion source was built and put into application in 1973, its high sensitivity attracted great attention, enabling, for instance, the surprising detection of nicotine in the blood of nonsmokers. At the same time, the group made an important contribution to the then arising public discussion about the risks of passive smoking. With the specific constructional peculiarity of the API ion source, first attempts at direct liquid chromatography (LC)–MS coupling became possible.

Figure 2: Hartmut Frank, Marjorie Horning, Ismet Dzidic, and Carol Howell in 2015.

Using the analytical methods developed, Marjorie studied the distribution and metabolism of drugs in the human body. Together with the perinatal physician Reba Hill, who also worked at Baylor College, Marjorie was able to prove in 1973 that drugs and even metabolites present in a pregnant woman's bloodstream could also enter the organism of the embryo or fetus. So far, the placenta had been considered a barrier, but further research showed that a variety of substances (drugs, nicotine, alcohol, and their degradation products) could pass through the placenta and reach the unborn child. The transmission of drugs to the baby via breast milk was demonstrated shortly thereafter, in 1975.

Thanks to all this pioneering work in which Marjorie was intensively involved, the Institute for Lipid Research became a leading center for the development of modern biomedical–analytical methods and a magnet for postdoctoral researchers from the United States, Japan, and Europe. For many, the work at the institute was influential for furthering their professional careers. The Hornings had created a stimulating work environment, highly attractive for young ambitious scientists, and, at the same time, full of lively social and cultural exchange. Marjorie and Evan complemented each other, both professionally and personally. Evan, while kind, was somewhat distant to his coworkers. Disciplined and serious in his scientific approach, he expected the same kind of conduct from his colleagues. Marjorie, however, was more affable and shared her joie de vivre with her coworkers when she came back from one of the many congresses to which she and Evan were invited. At the same time, the institute was an attraction and crossroads for leading international scientists and creative minds of the time, such as Arthur Karmen, Bo Holmstedt, and Robert Finnigan.

Marjorie Horning has been a member of editorial boards of several prestigious journals and was active in various US scientific societies and bodies. She was a member of the Society of Toxicology for many years, and was involved in the 1978 National Toxicology Program of the United States. From 1974 to 1983, she served on the board of the Southeastern Texas Section of the American Chemical Society (ACS) and, from 1980 to 1983, on the board of the Society for Experimental Biology and Medicine. She was U.S. delegate to the International Union of Pharmacology from 1983 to 1986. She was Treasurer of the American Society for Pharmacology and Experimental Therapeutics (ASPET) from 1980 to 1983, and served as the society's first female president from 1984 to 1986. A member of the American Association for the Advancement of Science, Marjorie has received numerous awards and honors, including an Honorary Doctorate from Goucher College (1977), the Garvan-Oil-Medal of the American Chemical Society (1977), and the ACS Award for Special Achievements of Chemistry Researchers. In 1985, she became National Honorary Member of Iota Sigma Pi (the National Honor Society for Women in Chemistry). In 1987, she received the Tswett Chromatography Medal, and in 1990, together with her husband, she was awarded the Frank H. Field and Joe L. Franklin Award in Mass Spectrometry of American Chemical Society.

Following her retirement in 1987, Marjorie found more time to pursue her passion for art. Both Hornings were enthusiastic art collectors and patrons; their numerous trips to Europe may have contributed to the fact that both were "infected at an early stage by the 'illness' of collecting." Their focus was on woodcuts and engravings from the old masters such as Dürer and Rembrandt, which they mainly acquired in Europe. Since 1974, they have donated more than 230 sheets from their collection to the Museum of Fine Arts Houston (MFAH), laying the foundation for the Department of Prints and Drawings at the MFAH. Some of these pieces can be viewed on-screen in a recent online exhibition "The Marjorie G. and Evan C. Horning Collection" with comments by Marjorie G. Horning (https://artsandculture.google.com/exhibit/lQJSACh9MqxILA).

Acknowledgment

The assistance of Dr. Ismet Dzidic in ensuring the accuracy of some dates and facts is greatly appreciated.

This article was originally part of a presentation by W. E. at the symposium "30 Years of Postgraduate Studies in Toxicology and Environmental Protection at the University of Leipzig" on March 23, 2018, in Leipzig, Germany.

References

(1) Chromatographia 23(9), 686 (1987).

Werner Engewald is with the University of Leipzig in Leipzig, Germany. Hartmut Frank, postdoc at the Institute for Lipid Research 1974-76, is with the University of Bayreuth in Bayreuth, Germany. Direct correspondence to: Hartmut.Frank@uni-bayreuth.de