In 1923, Otto Meyerhof and Archibald V. Hill received the Nobel Prize for work on the energetics of muscle metabolism, in particular for the discovery of the relationship between oxygen consumption and lactic acid metabolism. In the same year, Otto Warburg and Seigo Minami published the first observations on changes in the metabolism of tumors . They had observed that tumors acidified the Ringer solution (an isotonic salt solution, with 2.4 mM NaHCO3) when 13 mM glucose was added, as indicated by a change in the color of organic pH-indicators. In this acidified solution, lactic acid was chemically identified. To better quantify this phenomenon, Otto Warburg modified the Barcroft manometer to measure slices of a Flexner-Jobling rat hepatoma, which he had received from Rhoda Erdmann at the Rockefeller Institute. The amount of lactate produced was calculated from the increase in CO2-formation during a 30-min incubation period. Surprisingly, the tumor tissue had a 70-fold higher rate of lactate formation than the normal liver as well as kidney and heart tissue likewise tested. This is the observation that would more than 50 years later be referred to as the Warburg effect. Lactate production did not depend on the presence of oxygen. That had not been expected, since according to Pasteur, the presence of oxygen should have suppressed glycolysis. The fact that there appeared to be no direct relationship between respiration and glycolysis lead to the conclusion that in cancer cells, glycolysis was a reaction which could produce energy, independent of respiration (oxygen consumption). In other experiments with varying glucose and bicarbonate concentrations, it was shown that there was no generalizable difference in oxygen consumption between the tumor and the respective normal epithelial tissue . In 1924, Warburg hypothesized that there was a defect in the relationship between glycolysis and respiration. Even though this observation was corroborated with other tumors by several contemporary scientists , the observation that oxygen could not suppress glycolysis prompted him to propose that a damage in respiration leads to carcinogenesis . This came to be a highly controversial issue climaxing in his famous papers in Science in 1956 .
Testing the effects of other parameters, Warburg and coworkers changed the pH of the Ringer solution ranging from pH 7.83 to 6.66 using 1–15 % CO2-N2 gas mixtures, respectively. The rate of CO2-production (interpreted as glycolysis) increased with increasing alkaline pH. Moreover, a tenfold increase in bicarbonate concentration at a defined pH of 7.5 also increased CO2 production . Warburg interpreted these conditions as being similar to those in blood passing through capillaries, leading at the same time to a modest acidification and to an increase in bicarbonate concentration. In the balance, glycolysis of the tissues would not change. On the other hand, other studies showed that in tissue homogenates, alkalinity increased with dedifferentiation and necrosis of tumors , suggesting that the tumor itself may have a different pH. However, the influence of pH on the growth of tumor cells appeared never to be of particular interest to Warburg, in spite of his interest in hydrogen-transferring systems such as the coenzymes NAPDH and NADH (see below), which lead to the characterization of the activity of most glycolytic enzymes in later years.
Warburg corroborated his in vitro results in rats having either a hepatoma or sarcoma, where he found a higher lactic acid content (chemically determined) in blood vessels leaving the tumor than in vessels entering the tumors . Similar experiments had been performed by Carl and Gerty Cori , who also found different lactic acid levels in the blood of the two wings of same chicken: one with the implanted tumor and one without it. Warburg’s interpretation was that a lack of oxygen (hypoxia), along with an increase in lactic acid, favored the survival of tumors as opposed to normal cells, since the latter could not recruit their energy from anaerobic glycolysis. In other words, his hypothesis was that chronic hypoxia would damage respiration. The basis for this line of thinking was that, according to the Pasteur Effect, the presence of oxygen should (completely) suppress glycolysis. Since this was not the case in tumor cells, he concluded that there were “disturbances in the relationship between respiration and glycolysis” . In 1930, Warburg reinforced his hypothesis in stating that anaerobic glycolysis of tumor cells is the result of respiratory damage (Schädigung) . This issue was critically discussed by Dean Burk at a Cold Spring Harbor Symposium on Quantitative Biology in 1939, where he presented a collection of data from different tumors showing that tumor cells also displayed a Pasteur Effect since a fraction of glycolysis was indeed attenuated by oxygen, often to a similar extent as in normal growing cells .
Even though Warburg was also nominated for the Nobel Prize in 1926—for which he considered himself duly eligible—the Nobel committee decided to award it solely to Johannes Fibiger, honoring his findings on a gastric tissue growth condition believed to be a cancer induced by a nematode (spiroptera carcinoma). This has been considered as a misjudgment of the Nobel committee, as it later turned out not to be true [3, 16].
The respiratory ferment
Along with the basic questions on what kind of changes in energy metabolism convert normal cells to tumor cells, Warburg was interested in the chemical basis for the “respiratory ferment” responsible for oxygen transfer in cells. Warburg had already postulated in 1914 that iron had a catalytic function in cellular respiration. Also, Warburg knew David Keilin, who (in 1925) had spectroscopically detected three cytochromes with iron-containing porphyrins (hemins) in respiring cells. Since the available amount of the ferment was too small for analytical chemistry, Warburg applied an “inhibition technique” by using two substances having specific inhibitory effects: hydrocyanic acid and carbon monoxide (CO), the first inhibiting respiration irreversibly, the latter reversibly depending on O2-pressure . Visiting Warburg’s lab, Alan Hill brought to his attention that the inhibition of respiration by CO was light-sensitive. This allowed Warburg to characterize the oxygen-sensitive ferment by relating changes in the absorption coefficient determined by spectrometry with increases in the respiratory activity upon increasing the illumination . In this way, Warburg identified cytochrome a
(cytochrome oxidase) as being the CO-sensitive respiratory enzyme, i.e., the one requiring oxygen. Today, we know that there are indeed five proteins with iron for the electron transport and that cytochrome oxidase is part of complex IV. Warburg furthermore postulated the respiratory proteins to be localized in the “grana” of cells, which years later were identified as mitochondria.