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1 Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44094. Fax 440-256-3280; e-mail doc{at}arcwebserv.com
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Introduction |
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 Top Introduction References |
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I was 11 when the stock market crashed and the Great Depression began. Unemployment reached 25%. The most precious thing a person could have was a job. In 1940, I entered graduate school studying biochemistry at what is now Case Western Reserve University. Victor Myers was head of the department. He was near the end of his career and was prone to talk about his old friends Otto Folin and Stanley Benedict and others and their struggle to learn how to analyze blood. They were the first clinical chemists. They had no pH meter, no spectrophotometer. Optical densities were estimated by visual comparison of standard and unknown. They had to calibrate their own pipettes. It could not have been easy.
When I entered the department, most everyone used DuBosc visual colorimeters. We had one Evelyn photoelectric colorimeter with glass filters and one newly invented Beckman pH meter. There was no good way to determine the quantitative amino acid composition of proteins. No one understood what the purine and pyrimidine bases and nucleic acids were good for. The Journal of Biochemistry published one modest volume a year.
I do not think any of us understood that we were caught up in a wave of scientific and technological progress that would grow and gather speed at an exponential rate. I am glad to have lived during this period and to have been a very, very small part of it all.
By 1941, I had my Master’s degree and was working for my PhD. My sweetheart, Jean Hossel, was in her third year of nursing training at nearby St. Luke’s Hospital. We planned to get married the next year. Then suddenly, without warning, the Japanese struck at Pearl Harbor. It was on a Sunday in the early afternoon. I was in the Rat Room at the Cleveland Clinic doing vaginal smears on rats. That evening, while sitting in my 1936 Ford V8 coupe outside of St. Luke’s Hospital, Jean and I decided to marry without delay. We were married 5 days later, as soon as the license permitted, and Jean moved into my rented room with me because the rent had been paid.
Not long after that we were both at Ben Venue Laboratories, which had a contract to lyophilize blood plasma for the army. Jean was in charge of a group of nurses who made up pools of plasma and distributed them in transfusion bottles. The bottles were shell-frozen. I was given the responsibility of drying them from the frozen state. This was a new and little-known process. It was a very difficult time and a heavy responsibility until we finally learned the process.
By the spring of 1943, drying plasma had become a routine process. I persuaded my draft board to reclassify me from 2b to 1a and enlisted in the Navy. I was given orders to report to Officers’ Training School at Cornell in August.
In the intervening 3 months, I worked at the SMA Corporation on the purification of penicillin from culture medium, which consisted of corn steep liquor; a thick dark brown gooey liquid. All I remember now is that we extracted the penicillin into amyl acetate and back into water. I remember very clearly Dr. Paul Gyory storming into our laboratory one Saturday morning demanding penicillin. I gave him the dark brown solution I had just made. He took it to the hospital, gave it to his patient, and saved his life. This was a thrilling moment for me that I will never forget. The discovery of penicillin was perhaps the greatest discovery of the 20th century.
After Officers’ Training, I was ordered to the USS Hovey, DMS11, a World War I destroyer converted to minesweeper duty. I served as Gunnery Officer until we were torpedoed and lost our ship in the Lingayen Gulf in the Philippines.
The war over, I went back to school on the GI bill and with my
mother’s help. Victor Myers died, and Jack Leonards was appointed
acting chairman of the department. Jack came in the laboratory one day
in an excited state. He had a monograph written by Willem Kolff, a
Dutch physician who had invented an artificial kidney (Fig. 1
) that actually kept uremic patients alive by dialysis. It
consisted of a rotating drum partially immersed in a big tub of a salt
solution around which cellophane sausage tubing had been wrapped. Blood
was taken from the brachial artery and propelled through the tubing,
using the principle of Archimedes’ screw, and then returned to the
vein, largely cleared of urea and unknown toxic substances.
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I thought we could do better. Jack liked my idea, took departmental
funds committed to other projects, and had our first kidney made by
Sieberling Latex Products. It consisted of several units, usually 12,
each consisting of a pair of molded rubber pads with two sheets of
cellophane between them (Fig. 2
). The patient’s blood was propelled by a pump between the
cellophane sheets while dialyzing solution ran on both sides between
the cellophane and the rubber pads. The urea clearance varied between
60 and 80 mL of blood cleared per minute; we could return the blood of
uremic patients back to near normal within 6 to 8 h. I remember
one comatose patient that we treated who after treatment demanded steak
for breakfast. Our kidney was the first flat-plate kidney
(1) and was an important step in the development of today’s
artificial kidneys.
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After obtaining my PhD, I took a job as head of the Clinical Chemistry Laboratory at the Veterans Administration Hospital associated with the Case Western Reserve University Medical School. My boss was Joseph Kahn, pathologist and chief of the clinical laboratories. Joe had worked with Harry Goldblatt, who had shown that hypertension could be produced in animals by reduction of the blood flow to the kidney (2). He also showed that the increase in blood pressure was attributable to the liberation of an unknown pressor substance into the bloodstream (3). This was a discovery of the first order, being the starting point in the development of our present understanding of high blood pressure. It should be understood that at that time there was no good way to control the blood pressure of hypertensives, and as a consequence, many died of stroke or kidney and/or heart failure. Many young men and women in their 20s and 30s contracted malignant hypertension with very high, uncontrollable blood pressure and died. Harry Goldblatt should have received the Nobel Prize but did not.
The most important question among those who were interested in or working in hypertension at that time was the identity of the pressor substance from the ischemic kidney. It was known that extracts of kidney did contain a pressor substance called renin, but many thought that renin could not be the causative agent in hypertension because it was tachyphylactic. That is, after several doses were given to animals, it no longer raised blood pressure.
Irvine Page and Oscar Helmer (4) in this country and Eduardo Braun-Menendez and his group (5) from Argentina simultaneously discovered that kidney extracts containing renin when incubated with blood plasma produced a heat stable, dialyzable, pressor substance, now called angiotensin.
Joe Kahn and I, with no research funds, borrowed time from our other
duties and equipment and material from everyone we could and decided to
look for angiotensin in the dialysates of the blood of hypertensive
dogs (Fig. 3
). Using the artificial kidney, we equilibrated the dogs’ blood
against large volumes of dialyzing solution for 90-min periods. We
worked out a method of purifying the dialysate, reducing its volume
from 300 mL to 1 mL. This small volume was assayed in rats (Fig. 4
). The results are tabulated in Table 1
and show significant amounts of angiotensin in the dialysate of
hypertensive dogs and almost none in normotensive dogs
(6)(7).
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This was the beginning of our work in experimental hypertension, which
continued until my retirement in 1988. Joe and I were soon joined by
Walter Marsh who left us and was replaced by Kenneth Lentz. Still
later, Harry Hochstrasser and Frederick Dorer became members of our
group. Ann Gould and Melvin Levine were also with us for a time. My
good friend Joe Kahn worked with me until he retired shortly before I
did. The accomplishments of our group can be understood by reference to
a simplified diagram of the renin angiotensin system shown in Fig. 5
.
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We set out to purify angiotensin. We soon learned that there were actually two forms of the peptide, angiotensin I and II (8). We isolated angiotensin I in pure form and determined its amino acid concentration (9)(10). We purified angiotensin II and determined its amino acid composition and sequence (11)(12). We discovered that angiotensin I is converted to angiotensin II by the angiotensin-converting enzyme (13), which we partially purified and found that it was activated by chloride ions (14).
We also found that angiotensin I has no effect on blood pressure, whereas angiotensin II molecule-for-molecule is perhaps the most powerful pressor agent known (14).
We purified renin substrate from hog plasma and found that it had several different forms (15). Subsequently, we degraded the substrate molecule with trypsin and obtained a peptide substrate. The peptide was isolated from the digest, and its structure was determined and found to be that of a tetradecapeptide (16). The structure was confirmed by synthesis, yielding a fully active molecule (17). Finally, nine different peptides were synthesized that represented portions of the peptide molecule, and the kinetics of their reaction with renin were determined (18).
Since then, inhibitors of the converting enzyme have been developed that effectively block the renin-angiotensin system in vivo and are widely used to control high blood pressure and to decrease the after load on the heart in congestive heart failure.
While I was deep into research on hypertension, I was also being paid to run the Chemistry Laboratory for a 1000-bed hospital. I had three, sometimes four, technicians who also collected all the blood samples, washed and sterilized their glass syringes, sharpened and sterilized their own needles, and washed their own glassware. We did have glass filter colorimeters, which were just then replacing the old visual DuBosque colorimeter. Carbon dioxide combining power was done with Van Slyke’s manometric apparatus, with frequent mercury spills that ended up in the cracks of a wooden floor. Each day, my technicians had hundreds of manual operations to perform while talking to each other about last night’s date, the baseball game, or just polishing their white shoes. I had one very good male technician, Al Nagy. He was the only one I ever knew who could smoke a pipe and pipette at the same time.
I was worried about the quality of the results. I put unknowns into every batch of analyses and found frequent, very bad errors. There were just too many manual operations. I dreamed of a machine that would do analyses without error.
One day, it suddenly occurred to me that analyses could be done in a
continuously flowing stream rather than batchwise or discreetly. I told
Joe Kahn what I was thinking. He urged me to build such a machine and
loaned me the money that was needed to get started. I could not resist
his offer. And that was just the beginning. In all, Joe loaned me about
$5000, of which $3500 went for lawyers’ bills. Without Joe’s
financial and moral support, I would not have made the AutoAnalyzer. I
bought what was necessary and built model I, working in my basement at
home on nights and weekends. The result was the prototype shown in Fig. 6
. The small bottles at the right are urea calibrators; the large
bottles contain reagents. A peristaltic finger pump is just to the
left. Polyethylene tubing runs from the pump to mixing coils, a
dialyzer, and Coleman colorimeter fitted with a flow cell made from a
bent pipette. I fed samples by hand into the machine and read and
recorded the colorimeter every 15 s. After graphing the
colorimeter readings, I knew my idea would work. I had the proof of
concept.
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Model II is shown in Fig. 7
. The method, calibrators, reagents, and pump and dialyzer were
the same as in Model I. The tubing was 0.030-inch diameter
polyethylene. The grooves in the dialyzer were square, 0.030 x
0.030 inches. The colorimeter was my own design with a 6 V incandescent
light source powered by a constant voltage transformer with a glass
filter. The flow cell was 0.020 inches in diameter x 2 cm long.
The detector was a photomultiplier tube. The output was fed into a 10
mV industrial potentiometric recorder.
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At the outset I made every effort to prevent air from getting into the flowing stream. When changing samples I was careful to pinch the aspirating tube to prevent air from being introduced. Occasionally I did not get the tubing pinched and an air bubble would get in between samples. Of course, I noticed that the separation of samples was much better with the air bubble than without. Thereafter I always introduced air between samples and soon was adding air during sampling and in the dialysate stream as well.
The flow diagram of Fig. 8
contains my adaptation of a well-known urea method in which I
substituted a dialysate for a protein-free filtrate. The sample stream
was incubated with urease; the liberated ammonia entered the stream of
dialysate, which was nesslerized and passed through the colorimeter.
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A typical recording (Fig. 9
) shows 90-s samples of four urea calibrators with increasing
and decreasing concentrations, together with their optical densities.
Although a steady state was attained quickly, the record is noisy. I
simply drew a line through the peaks to estimate an average peak value.
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Typical results are shown in Table 2
and are satisfactory when compared with those obtained with
manual urease-aeration-titration method, which was the best method
available for urea in those days. The closeness of the higher values is
striking.
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At this point I licensed my machine to a small but reputable manufacturer of laboratory equipment. I sent him model II, together with my sketches and notes. Unfortunately, he thought I would furnish him with drawings for a finished marketable product, whereas I thought his company would engineer the final product. After several months we parted company. I got back a beautiful but empty stainless steel cabinet and a box full of my parts.
At this point I was very discouraged and was going to quit and put the
box of parts up in the attic. Jean said, "No". She would not let me
give up. So I built model III (Fig. 10
). Many of the parts were the same. There was a new automatic
sampler, a new dialyzer, and a colorimeter and heating bath. Model III
was capable of determining urea and also glucose, using ferricyanide
and inverse colorimetry.
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I then built model IV in an attempt to further improve the machine. The
result is shown in Fig. 11
. It featured still another colorimeter of my design. The
colorimeter cover is shown at the left. The sampler from model III was
used but is not shown. Model IV is now in the Smithsonian Institute.
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It was during this time that I tried very hard to find a company to further develop, manufacture, and market my analyzer. Several companies turned me down. It seemed that no one wanted it. I told Jean that if I had a gold brick, I could not sell it because no one would believe it was gold. It was very discouraging. Finally, there was one person who had a very small company with few resources who offered me a contract. Jean and I talked it over and decided that I had better sign the contract. That was on a Friday morning. I put the contract in my pocket, ready to mail, and went to work. Later that day, a salesman from Technicon, Ray Roesh by name, came into the laboratory. He had heard of the machine and wanted me to bring it to New York on Monday. He insisted; I could not get rid of him. He even followed me into the men’s room. So I finally agreed. The contract in my pocket was forgotten.
Jean and I loaded the analyzer into our 1949 Ford, dropped our young daughter Laura off with her grandparents, and headed for New York. They put us up at the Waldorf. Monday morning we drove to the Bronx. We took a freight elevator to the sixth floor of a warehouse where Technicon was located. Jean and I set up the machine, drew blood from each other, and put it in the sampler. I did not tell them anything. As the peaks appeared on the recorder, Andy Ferrari and the others started to tell me how to build it. I signed a contract with Technicon several months later.
At that time Technicon was owned by Edwin C. Weiskopf, whose family came from Germany where they made thermometers. Mr. Weiskopf died in 1968, and his son Jack Whitehead inherited the company. Their principal product when I first met them was an automatic tissue staining device invented by Harry Goldblatt. They were good people who strove to make a product they could be proud of. I never had an argument with them. It is much to Jack’s credit that he founded the Whitehead Biomedical Institute with a large part of his estate.
Technicon spent 3 years engineering and developing the first single-channel AutoAnalyzer, I described the method and Technicon’s first AutoAnalyzer in 1957 (19). The first 10 analyzers sold for $3200 each and went to 10 selected laboratories across the country. I thought it was so expensive no one would buy it; I was wrong. Technicon soon had many orders to fill. It was an instant success.
Within a year the Coleman Company brought out an analyzer that was a clear infringement of my patents. Technicon sued Coleman. The trial was in Federal court in Chicago. I was on the witness stand for 3 days. It was very difficult. I went to the sanctuary in the Downtown Temple at the top of one of Chicago’s skyscrapers every morning to ask for strength for the coming day. Technicon won on all four counts of infringement.
Technicon’s sales mounted, and soon many laboratories across the country had one or more AutoAnalyzers, including my own laboratory.
I soon realized that although one might have several single-channel
AutoAnalyzers, there were many manual operations to do and many
opportunities for error, particularly in calculation of results and the
handling of data (Table 3
). What was needed was a machine that would do all of the
commonly ordered analyses on one sample, calculate the results, and
record them on one piece of paper.
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I built the first model of such a machine in my basement in the
evenings and weekends with the help of Harry Hochstrasser. Harry also
worked with me in our Veterans Administration hypertension laboratory.
Fig. 12
shows the sampler: four pumps, a dialyzer, a 95 °C bath,
flame photometer, and multiple colorimeter as viewed from above.
Recorders are not shown. Another view (Fig. 13
) shows the primary recorder, calibration equipment, and a
second recorder that presented the analytical results.
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The flow diagram is shown in Fig. 14
. Albumin was determined directly on diluted serum using HABA
dye. The rest of the sample went through a dialyzer, and the
nondialyzable stream was used to determine total protein and
CO2. The dialysate was used to measure glucose
and urea as well as chloride, sodium, and potassium by flame
photometer. The colored streams were hydraulically phased with delay
coils so that peaks from a given sample arrived at the colorimeter in
sequence and were recorded in sequence as shown in graphically
calibrated form in Fig. 15
.
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This first multiple analyzer was described in Clinical
Chemistry in 1964 (20). It yielded excellent analytical
results but ran only 20 samples per hour. It did prove the concept and
led to the very successful SMA 12/60 (Fig. 16
). It was a workhorse. In my own laboratory, serving a 1000-bed
hospital, the 12/60 with one operator handled most of the work of the
laboratory in 4–6 h. A typical SMA 12/60 record is shown in Fig. 17
.
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The 12/60 was the first analyzer to use controlled, regular bubble introduction, which reduced noise and permitted a 60-sample per hour rate. This was the work of Bill Smythe at Technicon and was a great improvement. It did not use an integrated flow diagram. Analytical streams were independent from each other but were hydraulically phased.
The culmination of the series was the SMAC, Sequential Multiple
Analyzer Computer, which performed 20 analyses on every sample every
20 s (Fig. 18
). The computer made hydraulic phasing unnecessary and
simplified calculation and printing of results.
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The SMAC was a very expensive machine, costing over $200 000. However, in a laboratory with a large number of samples per day the cost per sample could drop as low as $2 or $3 per sample, or 10 to 15 cents per analysis. In the 1940s, the cost of a single blood sugar was in the order of $5 or $6.
The SMAC was very successful and was sold all over the world. Eventually, it was criticized for its lack of selectivity. Critics said that it was wasteful and used too many reagents to do analyses for anything more than the physician really needed. My own view was that it was much less expensive to do 20 different analyses on every sample, which nearly always included all of the tests physicians needed, than to sort out all of the samples and perform only those analyses that physicians actually ordered. Moreover, early work by Ralph Thiers and his group had shown that a multiple analysis often gave the physician unexpected and valuable information (21).
Technicon, however, was sensitive to the wishes of the marketplace and modified the SMAC so that the operator could enter only the analyses that were requested and only these results would be printed. All of the results from the other analytical channels were there, of course, but were not printed and were discarded! This ensured that the requesting physician was not troubled by any unexpected but important abnormality in the whole 20-channel analysis.
Technicon attempted to modify the SMAC so that it would actually only perform those analyses on an individual sample that were requested by the operator. Although they made a major effort, spending a great deal of money, the project failed. This would have been the ultimate machine. Perhaps they gave up too soon.
It was not long after this that Jack Whitehead sold Technicon to Revlon and I no longer visited Technicon.
My own contribution to continuous flow analysis, other than as a consultant, ended with my development of the first SMA in 1964. Most all of the major findings of my laboratory in the VA hospital in the field of hypertension, listed above, were also made before this time.
By the mid 1970s, it had been well established that the blood pressure
of most hypertensive patients was attributable to the enzyme renin,
which could be assayed in their blood (see Fig. 5
). However, there was
and still is a smaller but very significant group of hypertensive
patients who have little or no renin in their blood. I thought it was
important to discover the mechanism that raised their blood pressure. I
worked on this problem with Joe Kahn, Ken Lentz, and Rick Dorer at the
VA laboratory until my retirement in 1982 and then went to work with my
good assistant Roseann Eadie (Fig. 16
) in the Biochemistry Department
at the Medical School.
After 6 more years, I finally reached the conclusion that low renin hypertension is caused in some mysterious way by renin or an altered form of renin without there being any significant amount of renin in the blood (22). I decided further that I would need a whole new lifetime to solve the problem and retired for the second time in 1988.
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Footnotes |
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References |
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TopIntroductionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  E. G. Erdos The ACE and I: how ACE inhibitors came to be FASEB J, June 1, 2006; 20(8): 1034 - 1038. [Full Text] [PDF]
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 N. L. Anderson and N. G. Anderson The Human Plasma Proteome: History, Character, and Diagnostic Prospects Mol. Cell. Proteomics, November 1, 2002; 1(11): 845 - 867. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) through mixing coils in an
incubating bath where ammonium hydroxide was produced from urea. The
mixture was passed through the dialyzer and discarded. Ammonium
hydroxide dialyzed into the stream of air-segmented water, was mixed
with Nessler’s reagent, debubbled, and passed through the colorimeter
flow cell, producing a recording as shown in Fig. 9
