Part 2

The other great advance in the use of phosphatic plant nutrients started with Liebig's recommendation (1840) to treat bones with sulfuric acid for solubilization. This idea was not entirely new; since 1832, a production of a "superphosphate" from bones and sulfuric acid had been in progress at Prague. At Rothamsted in 1842, John Bennet Lawes obtained a patent on the manufacture of superphosphate. Other manufactures in England followed and were successful, although James Muspratt (1793-1886) at Newton lost much time and "some thousands of pounds" on Liebig's idea of a "mineral manure."

It was difficult enough to establish the efficacy of bones and artificially produced phosphates in promoting the growth of plants under special conditions of soils and climate; therefore, the question as to the action of phosphates in the growing plant was not even seriously formulated at that time. The beneficial effects were obvious enough to increase the use of phosphates as plant nutrients and to call for new sources of supply. Active developments of phosphate mining and treating started in South Carolina in 1867, and in Florida in 1888.[25]

In a reciprocal action, more phosphate application to soils stimulated increasing research on the conditions and reactions obtaining in the complex and varying compositions called soil. The findings of bacteriologists made it clear that physics and chemistry had to be amplified by biology for a real understanding of fertilizer effects. After 1900, for example, Julius Stoklasa (1857-1936) pointed out that bacterial action in soil solubilizes water-insoluble phosphates and makes them available to the plants.[26]

The insight into the importance of phosphorus in organisms, especially since Liebig's time, is reflected in the work of Friedrich Nietzsche (1844-1900). This "re-valuator of all values" who modestly said of himself: "I am dynamite!" once explained the human temperaments as caused by the inorganic salts they contain: "The differences in temperament are perhaps caused more by the different distribution and quantities of the inorganic salts than by everything else. Bilious people have too little sodium sulfate, the melancholics are lacking in potassium sulfate and phosphate; too little calcium phosphate in the phlegmatics. Courageous natures have an excess of iron phosphate." (See volume 12 of _Nietzsche's Works_, edit. Naumann-Kroener, Leipzig, 1886.) In this strange association of inorganic salts with human temperaments, the role of iron phosphate as a producer of courage is particularly interesting. What would a modern philosopher conclude if he followed the development of insight into the composition and function of complex phosphate compounds in organisms?

From Inorganic to Organic Phosphates

By the middle of the 19th century, the source of phosphorus in natural phosphates and the chemistry of its oxidation products had been established. The main difficulty that had to be overcome was that these oxidation products existed in so many forms, not only several stages of oxidation, but, in addition, aggregations and condensations of the phosphoric acids. Once the fundamental chemistry of these acids was elucidated, the attention of chemists and physiologists turned to the task of finding the actual state in which phosphorus compounds were present in the organisms. It had been a great advance when it had been shown that plants need phosphates in their soil. This led to the next question concerning the materials in the body of the plant for which phosphates were being used and into which they were incorporated. Similarly, the knowledge that animals attain their phosphates from the digested plant food called, in the next step of scientific inquiry, for information on the nature of phosphates produced from this source.

The method used in this inquiry was to subject anatomically separated parts of the organisms to chemical separations. The means for such separations had to be more gentle than the strong heat and destructive chemicals that had been considered adequate up to then. The interpretation of the new results naturally relied on the general advance of chemistry, the development of new methods for isolating substances of little stability, of new concepts concerning the arrangements of atoms in the molecules, and of new apparatus to measure their rates of change.

In the system of chemistry, as it developed in the first half of the 19th century, the new development can be characterized as the turn from inorganic to organic phosphates, from the substance of minerals and strong chemical interactions to the components in which phosphate groups remained combined with carbon-containing substances.

Phosphatides and Phosphagens

The important phosphorus compounds in organisms are much more complex than the simple salts, to which Nietzsche attributed such influence on man's character. Long before he wrote, it was known that phosphoric acid combines not only with inorganic bases to form salts, but with alcohols to form esters. In the middle of the 19th century, Theophile Juste Pelouze (1807-1867) extended this knowledge to an ester of glycerol. This proved to be significant in several respects. Glycerol had been shown by Michel Chevreul (1786-1889) as the substance in fats that is released in the process of soap boiling, when the fatty acids are converted into their salts. That it has the nature of an alcohol had been demonstrated by Marcellin Berthelot. Instead of one "alcoholic" hydroxyl group, OH, like ethanol (the alcohol of fermentation), or two hydroxyl groups (like ethylene glycol), glycerol contains three such groups. It was the only "natural" alcohol known at that time. That this alcohol would combine with phosphoric acid could be predicted, but that the ester, as obtained by Pelouze, still contained free acidic functions and formed a water-soluble barium salt was a new experience.

ALCOHOLIC FERMENTATION

(C_{6}H_{10}O_{5})_{_n_} C_{6}H_{12}O_{6} C_{6}H_{12}O_{6} glycogen glucose fructose ^| ^| ^| || H_{3}PO_{4} || <-- ATP || <--ATP |v |v |v ---------------+ ------+ H--C--OPO_{3}H_{2}| H--C--OH | H _{2}C--OH | | | | | H--C--OH | H--C--OH | C--(OH)--+ | | | | | | HO--C--H O <==> HO--C--H O <=======> HO--C--H | | | | | | O H--C--OH | H--C--OH | H--C--OH | | | | | | | H--C--------------+ H--C-----+ H--C--------+ | | | CH_{2}OH H_{2}C--OPO_{3}H_{2}+ADP H_{2}C--OPO_{3}H_{2}+ADP

glucose-1-phosphate glucose-6-phosphate fructose-6-phosphate (Cori-ester) (Robison-ester) (Neuberg-ester) ^ | | | <-- ATP +----| | | +------| | | | v H_{2}C--OPO_{3}H_{2} | C(OH)--+ | | HO--C--H | fructose-1,6-diphosphate | O (Harden-Young-ester) H--C--OH | | | H--C------+ | H_{2}C--OPO_{3}H_{2} + ADP ^| || O || // CH_{2}OPO_{3}H_{2} || CH | |v | 3-phosphoglycer-aldehyde dihydroxyacetone-phosphate C=O <=============> CHOH (Fischer-ester) | | CH_{2}OH CH_{2}OPO_{3}H_{2} || + coenzyme + H_{3}PO_{4} O=C--OPO_{3}H_{2} | 1,3-diphosphoglyceric acid CHOH + dihydro-coenzyme (Negelein-ester) | CH_{2}OPO_{3}H ^| ADP --> || || O |v// C--OH | +---+ 3-phosphoglyceric acid CHOH + |ATP| (Nilsson-ester) | +---+ CH_{2}OPO_{3}H_{2} ^| |v COOH 2-phosphoglyceric acid | CHOPO_{3}H_{2} | CH_{2}OH ^| |v COOH | phosphopyruvic acid COPO_{3}H_{2} (enol-) || CH_2 ADP --> || COOH +------+ | +---+ |CO_{2}| + CH_3CHO <-------- C=O + |ATP| +------+ acetaldehyde | +---+ carbon | CH_{3} dioxide | + dihydro-coenzyme pyruvic acid | v +----------------+ | CH_{3}CH_{2}OH | + coenzyme +----------------+ ethyl alcohol

Shortly after this experience had been gained, it became valuable for understanding the chemical nature of a new substance extracted from a natural organ. This substance was named lecithin by its discoverer, Nicolas Theodore Gobley[27] (1811-1876), because he obtained it from egg yolk (in Greek, _lekidos_). He used ether and alcohol for this extraction. Had he used water and mineral acid instead, he would not have found lecithin, but only its components. As Gobley and, slightly later, Oscar Liebreich (1839-1908), subjected lecithin to treatment with boiling water and acid, they separated it into three parts. One of them was the glycerophosphoric acid of Pelouze, the second was the well-known stearic acid of Chevreul, but the third was somewhat mysterious. This third substance was the same as one previously noticed when nerves had been subjected to an extraction by boiling water and acid and, therefore, called nerve-substance or neurine. Adolf Friedrich Strecker (1822-1871) established the identity of this neurine with a product he had extracted from bile and which went under the name of choline. Adolphe Wurtz (1817-1884) succeeded in synthesizing this substance from ethylene oxide, CH_2.O.CH_2 and trimethylamine N(CH_3)_3.[28] Thus, all three parts were identified, and Strecker put them together to construct a chemical formula for lecithin, glycerophosphoric acid combined with a fatty acid and with choline (a hydrate of neurine).

{ OH } N { (CH_3)_3 } Choline { C_2H_4O }

C_18H_33O_2 } HO } } } PO C_16H_31O_2 } C_3H_5O }

Fatty Acids Glycerophosphate \--------v-------/ Lecithin according to Strecker

This formula was not quite correct. Richard Willstaetter showed that an internal neutralization takes place between the amino group and the free acidic residue. This is expressed in his lecithin formula of 1918.

CH_{2}.O.R | CH_{2}.O.R_2 | | O.CH_{2}.CH_{2} | / \ CH_{2}.O--P=O N(CH_{3})_{3} \ / \---O----/

When the aim was to distill elementary phosphorus out of an organic material, it did not matter whether this was fresh or putrified. For obtaining lecithin out of egg yolk and similar materials, it was essential to use it in fresh condition. Otherwise, enzymes would have decomposed it. Through more recent work, four enzymes have been separated, which act specifically in decomposing lecithin. Enzyme A removes one fatty acid and leaves a complex residue, called lysolecithin, intact. Enzyme B attacks this residue and splits off the remaining fatty acid group from it, enzyme C liberates only the choline from lecithin, and enzyme D opens lecithin at the ester bond between glycerol and phosphoric acid. This is shown in the following diagram.

ENZYMATIC SPLITTING OF LECITHINS

ENZYME SUBSTRATE PRODUCTS

A Lecithin Lysolecithin and fatty acids.

B Lysolecithin Glycero-phospho-choline and fatty acids.

C Lecithin Phosphatidic acid and choline.

D Lecithin Phosphoryl choline and diglyceride.

Several fatty acids can be present in lecithin from various sources: palmitic and oleic acid, besides the stearic acid which at first had been thought the only one involved. In another group of extracts from brain or nerve tissue, amino-ethanol H_{2}NCH_{2}CH_{2}OH is found instead of the choline of lecithin. The variations include the alcohol, to which the fatty acids and choline phosphate are attached, for example, glycerol can be replaced by the so-called meat-sugar, inositol, which has six hydroxyl groups in its hexagon-shaped molecule C_{6}H_{6}(OH)_{6}.

The generally similar behavior of these phosphate-and fat-containing substances was emphasized by Ludwig Thudichum (1829-1901). He coined the name phosphatides for this group of substances from seeds and nerves.[29] His work on the phosphates in brain substance aroused particular interest. When William Crookes drew his highly imaginative picture of an "evolution" of the chemical elements, he put into it "phosphorus for the brain, salt for the sea, clay for the solid earth...."[30] But phosphatides occur in many places of organisms, in bacteria, in leaves and roots of plants, in fat and tissues of animals. And where phosphatides are found, there are also enzymes that specifically act on them. They are called phosphatases to imply that they split the phosphatides. In addition, enzymes are present, which transfer phosphate groups from one compound to another. They are more abundant in seeds of high fat content than in the more starch-containing seeds, but even potatoes and orange juice have phosphatases.[31]

Thus, from phosphatides, phosphoric acid is generated, and they could also be called phosphagens. Since 1926, however, the name phosphagens has been reserved for a group of organic substances that release their phosphoric acid very readily. The link between phosphorus and carbon is provided by oxygen in the phosphatides, by nitrogen in the phosphagens. In vertebrates, the basis for the phosphoric acid is creatine, whereas invertebrates have arginine instead.

H OH OH | / / N--P=O NH--P=O / \ / \ C=NH OH C=NH OH \ \ N--CH_{2}COOH NH | | CH_{3} CH_{2} | Creatine phosphate CH_{2} | CH_{2} | CHNH_{2} | COOH

Arginine phosphate

Nuclein and Nucleic Acids

All parts of an organism are essential for life. Only with this in mind does it make sense to say that the most important part of the cell is its nucleus. From the nuclei of cells in pus and in salmon sperm, Johann Friedrich Miescher (1811-1887) obtained a peculiar kind of substance, which he named nuclein (1868). Its phosphate content was easily discovered, but to find the exact proportions and the nature of the other components required special methods of separation from phosphatides and other proteins. It was difficult to develop such methods at a time when little was known about the properties, and particularly the stability, of a nuclein. For preparing nuclein from yeast cells, Felix Hoppe-Seyler (1825-1895) described the following details: Yeast is dispersed in water to extract soluble materials, like salts or sugars. After a few hours, the insoluble material is separated, washed once more with water, and then extracted with a very dilute solution of sodium hydroxide. The slightly alkaline solution, freed from insoluble residues, is slowly added to a weak hydrochloric acid. A precipitate forms which is separated by filtration, washed with dilute acid, then with cold alcohol, and finally extracted by boiling alcohol. The dried residue is the nuclein.[32] It contains six percent phosphorus. A little more washing with water, a slightly longer treatment with acid or alcohol gives products of lower phosphorus content. Many experimental variations were necessary to establish the procedure that leads to purification without alteration of the natural substance.