CHAPTER I.

REVIEW OF PREVIOUS INVESTIGATIONS.

Historical:

In 1836 Chevreul (222) found creatine in meat extract. The history of the investigations released by this discovery may be divided into three periods, first one from 1836 to the introduction of Folins (398) famous colorimetric method for quantitative determination in 1904, second one from that time to the discovery of the phosphocreatine by Eggleton and Eggleton (332) and Fiske and Subbarrow (384) in 1927, and third one from then to the present days.

The first period is chiefly taken up by chemical research and investigations on the occurrence of creatine and creatinine: The discovery of Chevreul was a t first rejected by Berzelius (89), but shortly afterwards confirmed by Schlossberger (1 185). In 1844 Pettenkofer (1068), corroborated by Heintz (542, 543), isolated from urine the substance which Liebig (728, 730, 731) in 1847 called creatinine, establishing its relations to creatine. The latter found both substances in human urine. In 1848 Schlossberger (1186) found creatine in human muscles. In 1849 Heintz (545) maintained that only creatinine is preformed in urine, but in 1858 Liebig (729) stated that the same may be the case withcreatine. Creatine was found in brain (926), smooth muscles (1234), blood (1379) and testis (1263, 1347). As a curiosity may be mentioned that Valenciennes and Fremy (1370) in 1855 suggested that creatinine in the muscles was an important substance, combined with phosphoric acid. For further observations on creatine bodies in muscles and urine of this period, see a).

a) 487, 490, 572, 634, 745, 907, 1252, 1253, 1433.

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The first attempt at quantitative determination was made by Staedler (1263) and carried on by Neubauer (976), whose method was again improved by Salkowski (1165, 1167), all of them using precipitation with zincchloride. A method invented by Kolisch (679), employing sublimate, proved worthless (486). On account of the imperfection of the methods quantitative estimations from this period are of little value - see a).

Much work was directed upon the physical and chemical proper- ties of creatine and creatinine, their compounds and disintegration products - these communications will be mentioned further on, in connection with remarks on chemistry. A curious little contest about the existence of one or more creatinines was settled in favour of the first view - b).

The second period was initiated by the results gained by Folin (389, 390) with his new method, the most important of which was his interpretation of urine creatinine as an exponent for the endo- genous metabolism. Now there followed a time of quantitative investigations. New methods were developed from Folins, for urine, blood and tissues. The relation of the creatine bodies to food, labour, sex, age, disease and several other conditions was examined, the search for the origin of creatine began. The results of these investigations form the chief subject of the following pages and will therefore not be referred to here.

As time went on the material accumulated, fact was added to fact without satisfactory explanation, theory to theory without definitive proof. The whole question was threatened by stagnation in an eddy of contradictions.

Eggletons (332) and Fiske and Subbarow (384), who were studying phosphoric acid in the muscles, discovered, independent of each other, that it formed a labile compound with creatine, important for the muscular contraction. In this way a distinct and significant metabolic task was attributed to creatine and a firm starting point established for the explanation of the facts already known.

During the third period the investigators have until now chiefly been occupied in elucidating the part played by the phosphocreatine

Then help came from another quarter.

a) 575, 845, 968, 969, 980, 1171, 1209, 1379. b) 632, 633, G80, 1086, 1318, 1344, 1433.

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in muscular metabolism. The corroboration of our earlier know- ledge with the recent discoveries has just begun, but may be expected to shed a new light on the creatine and ereatinine meta- bolism. An attempt in this direction is represented by this publi- cation ,

To the latest years belongs also the extensive literature on the relations of creatine in nervous diseases, called forth by the intro- duction of the glycine treatment, and contributing to the question of the origin of creatine. Furthermore the vivid interest now prevailing concerning the function of the endocrine glands has provoked several communications on the relation of these to the creatine and creatinine metabolism. These points will also be returned to later.

Physical and chemical properties:

anhydride of creatine: Creatine is methylguanidineacetic acid, creatinine is the internal

NH /

NHZ

HN = C / \

HN = C \

N(CH3) CH, COOH N(CH3) CHZ CO creatine creatinine

For investigations on the chemical constitution of the creatine bodies see a). Examinations of their disintegration products - b) and experiments on the synthesis of creatine also come in here. The synthesis was attempted with failure by Weltzien (1406) and Baumann (49), successfully by Strecker (1278 - sarcosine + cyan- amide), Vollhard (1380, 1381 - sarcosine + glycocyamine), Hor- baczewski (589 - sarcosine + guanidinecarbonate), Abderhalden and Sickel (9 - sarcosineethylester + guanidine), King (658 - thiocarbamidalkyliodide + sarcosine) and Bergmann and Zerwas (86, 87 - triacetylanhydroarginine + sarcosinemethy1- ester) .

For methods of purification seec).

a) 298, 346, 614, 730. b) 15,16,55, 188,269,297,298,426,472,635,730,736,792,978,1078,1419. c) 78, 262, 283, 312, 320, 321, 392, 398, 401, 403, 745, 783, 1270, 1305,

1322, 1327.

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Creatine has the molecular weight 131, is a colourless, well crystallizing substance, easily dissolved in hot water, with difficulty in cold water, very little is dissolved by alcohol. It is an ampholyt, the basic character being slightly predominating, - a).

Creatinine has the molecular weight 113, is also colourless and well crystallizing, the form of the crystals differs from that of creatine crystals. It is easily dissolved in cold water, and much easier in alcohol than creatine. It is distinctly basic - see a). Contrary to creatine i t is absorbed by charcoal, caoline and Fullers earth.

The transformation of creatine to creatinine and vice-versa is easily effected - this will be, mentioned later.

The compounds of creatine and creatinine are mainly salts, double-salts with metals, esters and substitution products, for the most part without interest for my work -see b). The one compound of dominating importance is of course the phosphocreatine. On account of its physiological significance I have treated i t separately later on, also from the chemical point of view. Of some importance is the alcohol of creatine, creatinol (1067, ll93), much more the double-salts of creatinine with potassium- and rubidium picrate and with zinc chloride, all used for precipitation methods (976, 484) and further the compounds giving the colour reactions.

For creatine Walpole (1387) gives a quantitative colorimetric method based upon the red colour given by creatine with diacetyl and alkali. Pittarelli (1072) ascribes to creatine a colour reaction with sodium nitroprusside and potassium persulfate.

For creatinine Weyl(l410) in 1878 found the reaction designated by his name with sodium nitroprusside and alkali, giving a red colour, turning yellow after some time. The reaction was studied nearer by Salkowski and others - see c). When acetic acid is added and the mixture heated the colour turns green and blue (1163). The reaction is not quite specific, thus it is given by acetone, according to McLean (777) the only difference being that, by

a) 320, 352, 511, 544, 841, 854, 1114, 1164, 1166, 1218, 1430. b) 255, 256, 303, 311, 345, 347, 480, 549, 550, 557, 612, 613, 623, 624,

643, 663, 681, 695, 787, 818, 826, 924, 976, 977, 979, 1086, 1112, 1189, 1190, 1281, 1365.

c) 96, 501, 691, 1164, 1336.

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adding acetic acid without heating after the changing from red to yellow, a straw colour is given by creatinine, a purple by acetone.

In 1886 Jaffe (624) discovered the reaction, which through the method of Folin (398) won a dominating position in the investiga- tion of the creatine bodies. It is produced by creatinine, picric acid and sodium hydroxyde, which give a pretty orange-red colour. Its strength is by Folin found proportional to the amount of creatinine, when the two other reagents are present in excess.

The chemistry of Jaffes reaction appears to be an intricate problem, to which has been dexoted much attention by Greenwald and others - see a), seemingly without final solution. The reaction is not specific, i t is given by acetone (624), H,S (398), glyco- cyamidine and hydantoinederivates (342), hydrolysed gelatins (892) and, assumedly, by unknown substances in blood, as will be men- tioned later. The disturbing effect of acetoacetic acid is spoken of in connection with the determination in the urine. Glucose will not disturb when i t is not heated (1403).

In the latest years new reactions are sought for. Komm and Leinbrock (671), Benedict and Behre (82) and Langley and Evans (706) describe a reaction with 3-5-dinitrobenzoic acid and alkali, giving a red colour, which is tried for colorimetric determination.

According to Reinwein (1103) the diazo reaction often ascribed t o creatinine depends upon impurities in the preparations.

The creatine bodies have a disturbing effect on the determina- tion of some other substances, namely guanidine derivates - b) and glucose. Creatinine is the strongest reducing agent in normal urine and influences the common sugar tests - c), in blood it disturbs the colorimetric glucose estimation (246, 495).

Occurrence:

The distribution of the creatine bodies in the animal kingdom is very interesting. The distribution of the phosphagens will also, for the sake of continuity, be mentioned here.

Creatine as phosphocreatine is found in all vertebrates in muscles, nervous system, gonads and in the electric organs of

a ) 30, 66, 473, 474, 477, 478, 470, 481, 483, 1172, 1453. b) 700, 769, 843, 1251, 1372, 1412. c) 350, 400, 777, 1423, 1432.

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for instance the Torpedo and the Raja. The presence of creatine and creatinine in blood is perhaps the most disputed question concerning these substances - see also further on. The existence of phosphocreatine in blood is hardly proved (10, 230, 582, 583). In urine there is always found creatinine, ordinarily little or no creatine, but more under special conditions. Phosphocreatine is not found there (10) - see a).

Between the different vertebrates there are some quantita- tive variations, for example the ruminants excrete considerable quantities of creatine (930, 1000) and in the urine of birds the creatine has taken the place of the creatinine, of which little is eliminated (846, 1054, 1328, 1378).

But passing to the invertebrates we find that the creatine bodies suddenly disappear, being replaced by arginine and possibly by agmatine (578), the phosphocreatine correspondingly by phospho- arginine and other, unknown phosphagenes (41, 44) - see b).

A few investigators have reported the occurrence of creatine in invertebrates, but these communications are mostly based upon colorimetric determinations alone and have not been confirmed, or have been directly disproved (128, 935, 973, 1122, 1370). There seems to be only one bridge over the gap: Phosphocreatine is found in the enteropneust Balanoglossus and in an echinoid (482, 972). It is extremely interesting that the way over echinoid and enteropneust is already, on morphological reasons, pointed out for the evolution from invertebrates to vertebrates by Bateson, McBrille and Garstang (quoted from 972).

Creatinine is reported to be present in soil (1231, 1283) and in vegetables (1341) and to be produced by several bacteria, is even proposed as a means for differentiation of these (31,455, 894, 1210, 1383). These communications, which are based upon colorimetric examinations, must be regarded with doubt since Linneweh (739)

a) Muscles: 39, 174, 235, 470, 490, 593, 596, 730, 766, 1033, 1038, 1149,

Nerves: 452, 926, 1294. Gonzds: 347, 906, 925, 974. Electric organs: 42, 43, 46, 659, 1201, 1411. Blood: 285, 290, 420, 936, 1179. Urine: 284, 409, 423, 1002, 1011, 1053, 1252, 1253, 1286. b) 17, 40, 128 129, 149, 330, 597, 699, 769, 862, 863, 902, 972, 1413, 1417,

1185, 1263, 1370, 1379, 1436, 1441.

1418.

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have failed to isolate creatinine from earth and plants in spite of positive Jaffes and Weyls reactions. In 1936 Solarino (1256) reports, that the colour given by bacteria on peptone medium originates from the medium, but that creatinine is possibly formed from bacteria on glucose medium.

Relations in urine-normal conditions, influence of sex and age: Since its introduction in 1904, the method of Folin (398) has

been practically (327, 328) the only one. The many mew)) methods published later - seea) can only be regarded as variations of the original one, this may even be said of the recent methods recommending 3-5-dinitrobenzoic acid as a substitute for picric acid (82, 671, 706).

The application of the autoclave for converting creatine to creatinine (Myers 71, 76), the introduction of creatinine as stan- dard instead of the original bichromate (Folin 392) and perhaps the employment of the Pulfrich photometer instead of the colori- meter (Kassel 647, Buhler 168, Lieb and Zacherl724) may be consi- dered as the most important improvements.

The colorimetric method, which has been controlled e. g. by Thompson (1327, 1332) has suffered remarkably little criticism. Linneweh and Linneweh (737, 738) found that in 10 % of the urine samples the value after autoclave treatment was 10-30 yo too high on account of unknown, ether-extractible substances. They were opposed by Stelzer (1269) although on small material, and as far as I know their attack has not been repeated and their view never confirmed.

The criticism of McCrudden and Sargent (838, 839) was called forth only by their impure picric acid (408, 601).

It seems, therefore, that Folins method for urine with its variants is one of the most recognized colorimetric methods we possess.

But, as mentioned before, the reaction of Jaffe is given or disturbed by certain substances other than creatinine. In urine the most important of these are the ketone bodies. Acetone augments the creatinine colour, 8-oxybutyric acid seems to be without effect, the action of he aceto-acetic acid is under discussion, by

a) 35, 76, 78, 79, 124, 163, 337, 369, 393, 510, 707, 718, 983, 1073.

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some maintained to augment the creatinine colour, by others to bleach it. Several methods have been invented to avoid errors on this account, some of them rather toilsome. The value for total creatinine is of course saved from disturbance of the ketone bodies by the heating procedure. Glucose under 5 yo is without effect. See a).

In 1905 Folin (390) declared that the diurnal elimination of creatinine on a diet without meat was individually constant, independent of the diuresis and total-N elimination and proportional t o the body weight when the fat was taken into consideration, 25 mg creatinine being excreted per kg in lean and 20 in fat indi- viduals. In putting forward his ))theory on protein metabolism)), he suggested that creatinine on a diet without meat might be regard- ed as an index of the endogenous metabolism (391).

His view was at once approved of by other investigators. Of these Shaffer (1221) maintained that creatinine was an index, not of the total endogenous metabolism, but of a certain part of it, >)the muscular efficiency)), i t was on the other hand not dependent of muscular work. For the creatinine elimination per kg of body weight per day he introduced the term ocreatinine coefficient)). Some confusion has arisen because he reckoned with creatinine-N instead of creatinine as Folin. The normal coefficient in the sense of Shaffer is therefore 5.4-11.7, that of Folin 14-26, both being used by different authors. See further b).

It was soon found, confirming an old observation of Hofmanns (575), that the creatinine coefficient was lower in women than in men - 14-22 against 20-26. The difference is ascribed to the different muscular development, the coefficient of well trained sporting women matching that of men (574, 1346). See c).

In children the coefficient and absolute quantity of creatinine eliminated is found to be low, rising from the birth to mature age - seed). Distinct racial differences have not been found (755, 1098).

From the introduction of the colorimetric method the normal

a) 95, 97, 203, 242, 306, 468, 471, 472, 475, 685, 908, 1138, 1427. b) 171, 238, 351, 409, 594, 828, 884, 1402. c) 75, 531, 686, 711, 840, 1041, 1221. d) 27, 28, 270, 325, 343,421, 438,443,444,489,641,754,778,798,1008,1125,

1133, 1152, 1153, 1169,1180, 1211, 1212, 1230,1292, 1350,1390, 1391, 1393.

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occurrence of creatine in the urine has also been the matter of interested examinations. Men on creatine- and creatinine-free diet are generally stated to excrete no creatine, though - according to Remen (1106) - a small quantity may be found.

Women under the same conditions eliminate small quantities, which are by some authors found to vary with the female sexual cyclus (686, 688), while others (923, 1134, 1265) deny this. Some women do not excrete creatine (1202).

Normal children excrete creatine from birth to the time of puberty. The exact age when the infantile creatine elimination ceases, or, in females, passes over in the adult form, is not quite clear (687, 732), some authors (642, 661) have a t times found creatine-free urine in younger children. The infantile creatinuria ceases abruptly, not by degrees (687).

The ototal creatinine coefficient)) - creatine + creatinine per kg per diem - is found to be like the adult creatinine coefficient (438, 531).

The cause of the creatine elimination in normal women and children is still quite obscure. Of the numerous explanations offe- red, the following may be mentioned: relations to the sexual glands - see later - to protein ingestion (271, 287, 291, 404, 435, 1393, denied in 1134, 1265), in children to undeyeloped muscles (1087), to thymus involution (771, 1031, 1177), to carbohydrate deficit (852, 1137, denied in 404, 1301). Thyreoid action seems disproved for children (1031), but possible in women (1202). See further a).

Blood: As recognized as the coIorimetric method is for urine, as bad is

its reputation for blood. At first i t seemed so easy to apply the urine method to blood

and serum and this was done by Folin himself and several others - see b) - with fairly good concurrence. The criticism of McCrudden and Sargent mentioned in connection with urine (839) was easily overcome, being occasioned by impure picric acid (408, 601).

a) 291, 293, 419, 439, 442, 558, 617, 687, 732, 733, 778, 1106,1137, 1301,

b) 80, 81, 115, 116, 124, 289, 358, 361, 367, 369, 395, 396, 406, 408, 410, 1367, 1391.

4ii , 418, 456, 485, 513, 522, 600, 673, 674, 825, 891, goo, 943, 1077, 1132, 1155, 1223, 1422, 1434.

2

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But in 1917 the first doubt came up with the work of Hunter and Campbell (601), who tried the colour development in relation to time and dilution and severely criticised all determinations of creatinine and of creatine.

The latter was found to vary only ))in a general waya with the real concentration. For plasma creatinine however the method was found to be fairly good.

After this time still more new improvements of the technique have been brought forward. The most recent of these are the methods for the Pulfrich photometer by Kessler (647) and especially by Lieb and Zacherl (724, 725). In 1934 Folin (399) gave the latest modification from his institute, using the colorimeter.

The methods have been constantly employed. In general the normal values 'are found to be about 1-2 mg %

for creatinine in serum and whole blood, this substance being equally distributed between plasma and corpuscles. For creatine in serum are mentioned values about 2 mg yo, but with wide variations. In whole blood is found about 5 mg yo creatine, this substance is thus supposed to be for the most part confined t o the corpuscles, where the value is estimated to about 8 mg yo. See a).

In renal retention all values are found augmented, which is utilized for diagnostic and prognostic purposes - see later and b).

Abel, Rowntree and Turner (11) found that creatinine, unfortu- nately only colorimetrically determined, passed out of the blood of living animals by dialysis, and Achard, Levy and Potop (13, 14) also stated that the creatine bodies in blood are ultrafiltrable and therefore must exist in a free state unless when the concentration is very high.

But by the work of Hunter and Campbell (601) the doubt was sown.

Several investigators attempted to isolate the creatine bodies from the blood. In most of these experiments blood from renal

a) 113, 299, 356, 357, 358, 359, 360, 446, 457, 505, 521, 602, 629, 630, 638, 639, 645, 673, 708, 719, 844, 890, 937, 998, 1363. (See also pag. 16 d.)

b) 33, 34, 94, 116, 183, 186,_187, 216, 233, 234,263,280,362,368,417,420, 447, 461, 499, 506, 507, 508,,517, 559, 604, 606, 631, 709, 743, 753, 850, 875, 943, 949, 950, 951, 975, 1058, 1095, 1131, Y146, 1147, 1235, 1260, 1353, 1371, 1385, 1396.

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retention cases was employed, only in a few cases normal blood was used, (246, 425, 427, 431, 433, 720, 735).

The values obtained were considerably lower than those given by colorimetric determinations.

The divergencies led to a very animated discussion, which is still going on.

The occurrence of creatine and creatinine in blood in renal re- tention is accepted by all. Further it is universally admitted that unknown colourgiving substances are present in blood and serum and disturb the colorimetric estimations, and especially that far too high values are found for creatine in whole blood.

But here the concordance ends. The question is, whether normal blood contains creatine bodies

a t all or whether all colour produced by Jaffes reaction should be ascribed to foreign substances.

The occurrence of creatine under normal conditions is doubted by Wilson and Plass (1077, 1422), Folin (396) and Berglund (85), but assumed by critical investigators as Behre and Benedict (68).

It is, however, the creatinine which has been the chief subject of the discussion, a vigorous and tenacious contest, which always seems to remain undecided: After a communication, issued with evident satisfaction from one side and obviously intended as a knock-out blow to the adversaries there is a short period of recovery and then from the other side a report appears, built on new and still more ingenious arguments and equally convincing in the opposite direction, and so the same thing recommences.

For the most part the conclusions are based upon the relation of the creatinine by heating with sodium hydroxyde - ))Benedicts 1. test)) - by adsorption with caoline - ))Benedicts 2. test)) - Fullers earth ())Lloyds reagent))) and agents of the same kind or by similar trials.

In this way differences are found between the original screatin- ineo of blood and serum and admixed pure creatinine, the latter being destroyed by hydroxyde and taken up by the adsorbents more easily than the former.

According to the view, whose principal spokesmen are Behre and Benedict (68, 69) this proves that the colourgiving matter in normal blood is not creatinine. But the opposite party, Danielson

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(272), Fetk-o-Luzzi (366), Hayman, Johnston and Bender (536) and Zacherl and Lieb (1440) maintain that the differences only concern parts of the colourgiving material, while the rest acts like real creatinine.

Some are also of the opinion that the employed tests are faulty, thus i t is found that picric acid shaken with caoline gives deeper colour by Jaffes reaction than untreated (536) and that by heating blood filtrate with sodium hydroxyde new colourgiving matter is produced (272, 366).

This is supposed to explain why so little wreatinineo is removed by heating and adsorption procedures.

It might be expected that the question would be settled by iso- lation of creatinine from blood, but there seems to be littleinclination to accept the reports given to that effect. Bohn, Friedsam and Hahn (1 15), who formerly supported Behre and Benedict, declare, however, that they are convinced by the recent communication of Linneweh (735), reporting the isolation of creatinine from large quantities - 10 1. -of normal blood.

The difficulties are illustrated by the newest investigations of Gaebler. From an ultrafiltrate of blood he precipitated a substance, supposed to be creatinine, first as double salt with potassium picrate (428, 433 , later as the corresponding, even less soluble rubidium compound (429). But in the most recent publication by this author known to me (430) he reports of important differences between the isolated substance and creatinine. He concludes that i t is different from, but related to creatinine.

A somewhat unusual support for the supposed existence of creatinine in blood is delivered by Miller and Dubos (881), who have discovered two different microbes with special affinities to creatinine, both possessing the power of decomposing this substance. One of them especially is very specific in its action and this microbe is found to remove from blood corpuscles 50 yo of the material, which gives Jaffes reaction, from serum still more.

But, as will be seen, the whole question is unsettled as yet, and the final result of the discussion cannot be predicted.

In this connection the question of a renal threshold for the creatine bodies ought to be mentioned as the view of the investig- ator on this problem is mostly directed by his attitude to the above.

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Those who maintain the existence of creatine in normal blood are forced to assume a threshold to be able to explain the normal creatine-free urine in men. On the other side those, who deny the occurrence of creatine bodies in normal blood, will also as a rule reject the existence of a threshold.

Cope (250) finds by comparing plasma creatinine and urine creatinine after ingestion of creatinine that direct proportionality exists when a threshold for creatinine is supposed a t a concentration of 0.5 mg per 100 cm3 in plasma. (See also 309,.)

The decision of this question must also await more efficient methods.

An attempt in this direction is represented by the new methods of Komm and Leinbrock (671), Langley and Evans (706) and Benedict and Behre (82), employing 3-5-dinitrobenzoic acid instead of picric acid. The value of these methods is still unknown. Benedict and Behre express the opinion that they may supplement, but not replace the earlier methods.

Oiher body fluids: In the cerebrospinal fluid the values for the creatine bodies are

found lower than in serum. Straube (1276) mentions 0.6-1.4 mg % for creatinine, 0.46-1.87 mg yo for creatine, creatinine is by practically all authors stated to be about % of the serum value.

In the cerebrospinal fluid both creatine and creatinine are augmented by renal retention, though more slowly than in blood. See a).

The creatine bodies are further found in milk (294, 295, 296, 394), in the amniotic fluid (382, 1104, 1211, 1368, 1415), in the saliva (308, 909), in gastric juice (300, 579, 607), in sweat (1204) and in ascites, trans- and exudates (148, 1157), observations which are a t present of little importance.

Tissues : For total creatinine in tissues the colorimetric determination

method is found to be excellent, in fact it has met with practically

a) 148, 329, 348, 445, 722, 723, 825, 943, 948, 1101, 1157, 1277.

22

no criticism. Only Gerard and Tupikow (452) and Ciaccio (229) mention the possible existence of unknown colourgiving matter.

The contributions of the several other authors are for the most part smaller improvements developed out of the original technique.

Micro-methods have even been proposed for such minute quantities of tissue as 0.2g (126, 1036). See a).

As for creatinine the case is different, because its presence in tissues has not as yet been demonstrated with certainty. Smaller amounts are found by many authors, but the difficulty is to exclude that this creatinine is not formed during the process of examin. ation from the large quantities of creatine present.

As far as I am aware, the publications on creatinine in tissues are ti1 now limited to mere statements of its presence or absence and no attempts have been made to account for its nearer relations. See b).

Some other earlier communications on the creatine bodies in tissues have been mentioned in my remarks on history and occurr- ence.

Beyond comparison the principal location of the creatine in the organism is in the striated muscles. Burger (171) finds by corrobor- ating the values given by Janney and Blatherwick (626) for creatine in the various organs and the tables of Vierordt for their relative weights, that 112 g of total creatinine are located in the muscles, only 2-3 g in other organs.

The quantity of creatine per weight unit of muscle is commonly stated to be about 400 mg yo in adult mammals, higher in quickly contracting muscles than in slowly contracting ones. See c).

The contents are much lower in embryos and somewhat lower in quite young animals, but will soon reach the mature value (210, 286, 848, 851, 938, 1137).

The corresponding value for creatinine is reported to be about 5-10mg % - see also b).

The dialysis of the creatine from the muscles, studied by Urano (1364) and Tiegs (1337), has gained special interest in the light of

a) 50, 51, 54, 179, 232, 240, 370, 394, 515, 527, 626, 814, 938, 942, 995,

b) 29, 160, 228, 848, 945, 980, 1171, 1209, 1223, 1225. c) 21, 22, 23, 70, 127, 225, 286, 402, 407, 422, 515, 620, 938, 967, 1022,

1115, 1116, 1145, 1223, 1398.

1025, 1135, 1188, 1223, 1242, 1246, 1248, 1437.

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the recent investigations on phosphocreatine and will be mentioned in connection with this.

Conditions which are injurious to the muscle tissue will as a rule lower the creatine contents, e.g. division of nerves (36, 201, 563, 1062, 1398) and nutritional dystrophia (462, 779, 984, 1377).

The creatine contents in striated muscles will be returned to later, namely in connection with labour, hunger and action of the endocrine glands.

In the cardiac muscle of mammals the creatine contents are found to be about 180-200 mg yo, higher in the powerful left ventricle than in the right one (1213). Here also the creatine contents are reduced through injurious processes, e.g. by infarctions (552). See a).

In unstriated muscles creatine is also found, in quantities slightly below 100mg %. See b).

Next to the muscles the nervous system, central and peripheral, is the most important location of creatine. Hammet (527) finds that the ratio of creatine to protein in the brain is 2 %, in the striated muscles 2.8 %. In the central nervous system the concentration is greatest in the cerebellum, next to this in the hemispheres. See c).

In the sexual glands, especially the testis, there are considerable creatine contents - see d).

The liver once played a prominent part in the discussion of the creatine and creatinine metabolism. I t was by some investigators presumed to be the organ where the creatine bodies were formed.

It was Mellanby (848) who first directed the attention to the liver. His chief arguments were that the appearance of creatine in embryonic life seemed to be closer connected to the development of the liver than of the muscles, and that liver diseases reduced the creatinine contents in urine, while some, as carcinoma of the liver, led to a large creatine elimination.

His theory met partly with approbation, partly with contra- diction. His suppositions concerning the embryonic development were challenged, but the interest was stimulated by the reports

a) 101, 160, 174, 248, 257, 259, 277, 278,279,422,552, 554, 555, 734, 1213. b) 160, 174, 1162, 1234. c) 452, 527, 530, 577, 814, 815, 820, 926, 1028, 1029, 1030, 1032, 1263. d) 1263, 1273, 1274, 1347.

24

of Hoogenhuyze and Verploegh (587), Gottlieb and Stangassinger (466) and others on autolysis and perfusion experiments on liver augmenting the total creatinine and the transformation of creatine to creatinine - see below page 40 and 43.

Also the remarkable increase of the creatine contents in the liver after ingestion of creatine, discovered by Chanutin (208, 211, 216, 217) pointed in the same direction. Chanutin himself a t first assigned a special importance for the creatine metabolism to the liver, but later concluded that i t was only a storage for the creatine.

The relations of the creatine bodies in the urine to the liver diseases have been differently interpreted, by some a connection is supposed, by others denied. Several authors have confirmed the reported creatine elimination in cancer of the liver. In one of the latest communications (Udeles and Shretter 1354) disturb- ances of the creatine metabolism by hepatic diseases is assumed, though on a dubious basis.

Experiments with liver extirpation and Ecks fistula show no relations between liver and creatine bodies.

In general it may be said, that nothing definite has yet been found proving any special connection between the liver and the creatine metabolism, and in recent years the interest in this question has abated without the attainment of a final solution. See a).

Creatine is also found in several other organs, but in insignificant quantities (132, 231, 313, 626, 644).

The relations of creatine in tissues are, of course, the part of the creatine metabolism which is most deeply affected by the recent discoveries of the significance of phosphocreatine, which will be mentioned later.

The examination of creatine in meat extracts forms a section for itself, with its own modifications of the methods. Attempts have been made to determine the quality and origin of the various commercial products by their creatine contents, but most of the authors find that no definite conclusions can be drawn on this basis. - See b).

a) 70, 208, 216, 217, 225, 365, 382, 415, 466, 558, 582, 583, 585, 587, 649, 713, 750, 762, 786, 797, 848, 852, 877, 882, 890, 939, 997, 1057, 1175, 1181, 1241, 1245, 1290, 1291, 1336, 1345, 1354, 1378, 1400.

b) 58, 59, 249, 344, 454, 488, 541, 671, 698, 764, 873, 874, 927, 928, 1069. 1247, 1248, 1249, 1250, 1270, 1280, 1375, 1395.

2 5

Labour and red: The abundant occurrence of creatine in the muscles naturally

suggested a connection between this substance and muscular work. The earlier investigations on this point were, however, disap- pointing, the changes found in the creatine metabolism during work were all insignificant or even dubious.

I t has been reserved for the investigation of the last ten years to prove that the connection guessed a t by earlier authors is a highly important reality. The processes, of which the phospho- creatine splitting is a link, are so rapid and well regulated that they were inaccessible to the methods of earlier times. The nature of these processes is also quite different from what was expected.

Much work has been spent upon examinations of the relations of the creatine bodies in urine to muscular work. While no increased elimination of creatine is found during work, most authors report a transitory increase in the creatinine excretion. For the most part it is found to be followed by a corresponding decrease and t o be of so short a duration that the diurnal creatinine elimination remains uninfluenced. See a),

The observation made by some authors, that more creatinine is eliminated per hour during the day than during sleep, has been explained as caused by the muscular movements. (181, 184, 185, 340, 662, 712, 1237.)

Most authors find that the creatine bodies in blood suffer no definite changes during work. (47, 448, 637, 1074, 1090, 1178, 1343.).

Regarding the creatine bodies in tissues, especially in the muscles, several publications exist, but in none of them there are reported any marked changes, only small irregular variations in both directions. By some investigators it is maintained that e.g. electrical stimulation increases the creatine contents of the muscles, by others the same effect has been found by central sympathetic irritation. See b).

Electric ,trainingo of muscles is found to augment their creatine contents (1015). The reduced creatine values in muscles after division of the nerves is mentioned on page 23.

a) 192, 200, 264, 340, 486, 489, 586, 588, 668, 746, 847, 872, 893, 964,

b) 154, 155, 165, 185, 241, 339, 422,654,963,1187,1203,1206,1208,1258, 1042, 1187, 1220, 1293, 1398, 1421.

282, 1331.

26

By Pekelharing (1061) it was assumed that the tonus of the muscles was related to the creatine bodies. As to this view a discussion arose, which, however, led t o nothing. In the first place this was owing to the confused interpretation of ))tonics muscular contraction. As examples were employed for instance the rigor after decerebration and contractures provoked by nicotine, veratrine and other chemical agents, but also phenomenons as the clasping of the male frog during copulation and even the straff military posture (I) See a).

Hunger: Even by earlier authors it was reported that the elimination of

creatinine decreased during starvation, a statement which later has been confirmed by most investigators. A few find the creatinine unaffected, only a couple of publicists maintain that the creatinine excretion may be elevated (636, 1261). See a) next page.

In 1907 F. G. Benedict (73,74) and independent of him Cathcart (197) discovered that creatine was eliminated during starvation. This discovery raised vivid interest and was universally accepted. Many of the values given must not, however, be considered as too trustworthy, as starvation is one of the conditions where ketone bodies may occur and exercise a disturbing effect on the estimation of the creatine bodies and very often the precautions necessary to avoid this error are omitted.

The possibility that no increased creatine excretion during starvation existed a t all, but that it was only simulated by the ketone bodies, was considered and rejected by Cathcart (203).

In general it may be said that sooner or later after starvation has set in, most often in a couple of days, the creatine appears or increases in the urine. By continued starvation the amount of creatine eliminated may remain constant (197), increase (852), or decrease slowly for some time and then increase again a t more advanced stages (592). The total creatinine may be unchanged (73) or slightly reduced (72).

Some attention has been bestowed on the terminal elevation, in animals has been found a so-called vcreatine-creatinine-crossing)), i. e. the quantity of creatine eliminated surpasses tha t of the

a) 319, 752, 885. 1022, 1061, 1062, 1115,1118, 1120,1192,1207,1267, 1369.

27

creatinine, This has been interpreted as being indicative of oncoming death (592), but may, according to others, occur even a long time before (903).

The readiness of starving individuals t o excrete creatine seems to be somewhat different, thus it may be found in some animals and wanting in others under the same conditions. See a).

The increased creatine excretion caused by hunger will abate immediately when food is given containing proteins and carbo- hydrates, not if it consists of fat only. See further on.

It is stated by practically all investigators that the creatine contents in the muscles augment during starvation.

This is a singular fact, especially when the loss of creatine in the urine is taken into consideration. Different explanations have been proffered: Other constituents of the muscles may be consumed more rapidly than the creatine, or the production of creatine may, for some reason or other, be increased, or the power of the organism to transform the creatine reduced. See b).

Several authors think that the hunger acidosis is the real cause of the creatine elimination - see further on under acidosis.

By comparing the amount of creatine and creatinine excreted and the contents of creatine in the body before and after a period of starvation Pariset and others claim to deliver a direct demonstra- tion of synthesis of creatine bodies in the organism, the sum of the lost and the remaining quantity being greater than the original one, provided that creatinine is formed from creatine (919, 939, 1043). See further origin of creatine.

In the brain the creatine contents remain unaffected by hunger (530, 1032), in blood the creatine bodies seem to be unchanged or a little increased (217, 418, 904), in other organs no definite varia- tions are found (217).

A cidosis : The frequent simultaneous occurrence of acidosis and increased

creatine elimination led several investigators to suppose a causal connection, the latter being regarded as a result of the former.

a) 131, 197, 489, 496, 586, 587, 590, 591, 598, 666, 713, 981, 1002, 1003, 1018, 1054, 1203, 1306, 1360, 1397, 1427, 1442, 1443, 1444.

b) 166, 215,,217, 219, 418, 470, 702, 853, 962.

28

In a still higher degree than when it concerns starvation, the possibility of errors caused by ketone bodies must here be kept in mind.

The evidences concerning this question are of two kinds. In the first place creatine is eliminated in considerable quan-

tity in diabetes mellitus, especially in acidotic patients, and the acidosis has been suggested as the factor producing hunger-creatin- uria.

Secondly numerous attempts have been made upon provoking or influencing the creatine elimination by administration of acids and acid, neutral and alcaline salts.

Low creatinine excretion in diabetes has been found long ago and this observation has later been generally confitmed - see a). Increased creatine elimination was reported by Krause (685) in non-acidotic cases, in acidotic ones by several others, and for a while the assumed connection acidosis-creatinuria occupied the attention - see also a), but soon discrepancies were met with, which called for another explanation.

Already by Burger and Machwitz (173) it was stated that, when the tolerance decreased, the creatine might appear before the ketone bodies.

This was corroborated by Lauritzen (710), who also found crea- tine before the acidosis, as measured by the ammonia elimination, and interpreted both as co-ordinate results of deficient carbo- hydrate metabolism. His conclusions were again supported by Brentano (144, 146), whose experiences will be dealt with more closely in the next section.

As t o the experiments with acids and salts no definite results were obtained - see b). Creatine elimination may sometimes be called forth and changes in the creatinine excretion noticed, but in these cases protein disintegration is increased and the increased creatine elimination is more probably connected with this than provoked by a special action of acids (434, 1312).

Riesser and Brentano (1 119) produced an artificial acidosis, controlled by pH-measurements, by administration of chlorammo- nia. They found that creatine was not constantly augmented and

a) 156, 203, 467, 703, 1146, 1275, 1303, 1358, 1423. b) 832, 833, 834, 1266, 1356, 6357.

29

not parallel to the acidosis and even that transitory creatine eleva- tion may be called forth by alcalosis.

Underhill and Baumann (1359) found crezitine-increase in hydrazine' poisoning when acidosis could be excluded, Palladin (1013) reported that in phlorizine poisoning the acidosis might be removed by administration of protein, while the augmented creatine elimination persisted.

I t may therefore be concluded that the assumed connection between acidosis and creatine in the urine is disproved.

Carbohydrates and creatine bodies: The examination of diabetes, acidosis and hunger-creatinuria

directed the attention to the carbohydrate metabolism and very interesting relations to the creatine bodies were found. The signs of these relations were for a long time vague, they were contended by a minority of authors who did not attain general recognition of their views.

But also on this point the recent achievements during the investigation of the phosphocreatine have brought new light of an engrossing nature and it may seem probable that this is a field where important discoveries are to be expected.

The evidence showing the connection between the Carbohydrates and the creatine bodies are heterogenous and suffer from the lack of a revision from a common point of view.

The relations of the creatine bodies in diabetes mellitus are mentioned in the precedent section.

During starvation the carbohydrates are found to possess great ability of abolishing the elevated creatine excretion. For the proteins this ability was a t first denied, later admitted, but by many authors ascribed to conversion to carbohydrate. Fats are found to increase still more the hunger-creatinuria. See a).

The poisoning with phlorizine has yielded interesting facts: As mentioned above the creatinuria was here proved to be indepen- dent of the acidosis. Cathcart and Taylor (204) found that the appearance and disappearance of the creatine augmentation as a rule coincided with that of the glucosuria, but the creatinuria

a) 199, 295, 852, 1013, 1054, 1139, 1266, 1425.

30

might be suppressed by ample administration of carbohydrate food while the glucosuria persisted.

Benedict and Osterberg (83) report that the elevated creatine elimination in phlorizinised animals will continue even if they are kept on positive nitrogen balance by abundant protein and they are of the opinion that such animals are ))diabetic)) in relation to creatine. Contrasting to this Lieben and Laszlo (727) have found no creatine elimination in phlorizinised animals in nitrogen balance and deny relations to carbohydrates.

Extirpation of the pancreas has the same effect as phlorizine poisoning (252, 1139).

The experiments of Underhill and Baumann (1359) on reducing the carbohydrate depots of the organism by hydrazine with result- ing creatinuria have already been mentioned. See also 768.

Similar attempts on carbohydrate depletion by sodium selenite also gave creatine elimination (202).

Carbohydrate deficit in the food is reported to provoke creatin- uria (1014) or no effect is found (468, see also 198).

A diet rich in carbohydrates will not do away with the infantile creatinuria (1301), but is reported to reduce the urine creatine in ruminants (1000). In contradiction to this i t is also reported (519) that glucose and fructose, especially in combination with work, may cause a creatinuria, but these experiments are not convincing.

The creatinuria discovered by Palladin in rabbits after cooling may according to his report be prevented by previous ad- ministration of food rich in carbohydrates (1012, 1014). Transitory increase of muscle creatine is found on a carbohydrate diet (940).

Augmented creatinuria is found coincident with hypoglycemia and with glycosuria (204, 727, 768, 1351, 1359). On the other hand most investigators assign to creatine the power of reducing the blood sugar and to reinforce the action of insuline (561, 675, 677, 1060, denied in 1262). Insuline is found to lower the blood creatine (676, 678, 1126).

Very interesting is the hypothesis advanced by Brentano (140, 141, 142, 143) that creatinuria is a consequence and sign of reduced glycogen contents in the muscles. He thinks he has found a reduction of the muscular glycogen under a diversity of conditions

31

where creatine elimination also occurs. Creatinuria connected with unchanged muscle glycogen he finds after having prevented the breakdown of the muscle carbohydrates by poisoning with sodium fluoride. He therefore has suggested that the creatine elimination is caused by reduced splitting of the muscular glycogen owing to deficit or obstruction.

To be able to transfer his investigations to man, Brentano has invented two indirect tests as substitutes for the direct examination of the glycogen contents of the muscles: After injection of adrenaline he finds in individuals with normal muscle glycogen a rise of the lactic acid concentration in blood, which is lacking when the glycogen is reduced and also lacks in individuals with marked creatine elimination (145).

As a supplement t o this he reports that in cases with creatinuria injections of adrenaline provoke an increase of ketone bodies in the blood, which is lacking in normal individuals (144, 146).

Querol and Reuther (1089) parily reject the results of Brentano, they find that the lactic acid elevation after adrenaline is absent in fresh cases of creatinuria, as reported by Brentano, but in cases of increased creatine elimination of longer duration they find it present.

But the experiments of Jahn (621,622) point in the same direction as those of Brentano. He reports that creatine injections besides hav- ing a tendency to lower the blood sugar also may diminish the lactic acid in blood, increase its contents of ketone bodies and reinforce the action of insuline. He assumes that the creatine supports the insuline in the building up of the muscle glycogen. In disorders of the creatine metabolism he suggests the occurrence of simulta- neous disturbances of the muscle glycogen, resulting in a relative insuline deficit caused by the increased requirements of the muscles.

Brentano and Jahn both deny any direct action of the creatine on the glycogen of the liver, but are forced to conceive some connec- tion between the muscles and the ketone body production of the liver.

As will be seen, these recent investigations approach from one side the same fields which have been entered from another quarter during the examinations of the phosphocreatine. I t may be expected that in near future contact will be established.

32

Adion of profeins: The power of counteracting the hunger-creatinuria undoubtedly

possessed by the proteins has been mentioned above - see page 27. The question of the influence of food proteins on the creatine

bodies in the urine has been emphasized with great interest. See a). Most authors assume that proteins ingested will increase the

quantity of creatinine excreted, even if the augmentation is not supposed to be very large.

As to the creatine there are considerable differences of opinion. Some authors say that the protein in food will increase the urine creatine, others deny this, a t any rate for moderate quantities. Recent authors find that the proteins of usual mixed food are without effect on the creatine, even if they increase the creatinine a little (339, 692, 1302, 1451).

But on the other hand, Terroine and his collaborators think that all creatinuria originates ip the catabolism of proteins, exo- genous or endogenous.

The creatinuria of children and women is by many investigators found to be augmented by food proteins, by some it is even as a whole ascribed to these. But other authors hold the opposite view - see b).

It is also maintained that the urine of males is creatine-free only when the diet does not contain proteins. But great individual differences are assumed, some are thought t o be able toingest more proteins without increase of the urine creatine than others.

The action of the proteins is by some authors ascribed to their .specific dynamic effect or to some process of irritation, but most investigators, who believe in the creatine-augmenting effect of the proteins, assume a direct conversion. This touches the question of the origin of the creatine bodies and will be returned to in due connection.

I t must, however, be remembered that in most of the protein experiments there are employed the proteins of milk, eggs and meat, articles of food which contain both creatine and creatinine.

a) 117, 164, 214, 226, 288, 301, 302, 339, 397, 436, 437, 459, 497, 531, 532, 533, 664, 665, 692, 798, 830, 831, 833, 835, 929, 1021, 1079, 1094, 1099, 1100, 1130, 1142, 1143, 1266, 1302, 1306, 1307, 1308, 1309, 1310, 1311, 1312,1313, 1314, 1316, 1339, 1451, 1455.

b) 271,291,292,293,434,435,440, 441,798, 1265,1391, 1393.

33

It is difficult, if not impossible to provide a sufficient diet, which is with certainty free from creatine bodies.

As a rule, therefore, the possibility cannot be excluded that the rise of the creatine bodies in the urine after ingestion of proteins is not due to their contents of creatine or creatinine.

As for meat, which has not been specially treated, we certainly must reckon with considerable quantities of creatine bodies in it. This has also been acknowledged by most investigators previously, meat has even been employed instead of pure creatine for testing the tolerance for this substance. The communications mentioned before on the effect of a mixed diet refer to the ordinary meat pro- teins and their contents of creatine bodies are taken into consider- ation. For literature see page 32 a).

Informations on the action of protein on the creatine bodies in tissues and blood are scarce, in blood augmentation has been found (640, 952, 1243, 1392).

As will be seen this question is one of the many concerning the metabolism of the creatine bodies, which has hitherto been solved contradictory and inconclusively. It is very difficult to arrange the experiments sufficiently simple and univalent to allow any decision and it is probable that this question must await the solution of that obscure and extensively discussed problem - the origin of the creatine.

Pregnancy:

One of the most puzzling conditions under which the creatine elimination is found to be increasing is pregnancy.

The creatinine elimination seems to be on the whole unalte- red, even if minor increase or decrease are reported by some authors.

The creatinuria in pregnancy is a common, but not constant occurrence. The quantity eliminated augments slowly towards the delivery, has a maximum shortly after it and then abates quickly in 12-14 days (1202).

The explanation of this phenomenon is not easy and has not yet been set forward in a satisfactory way, in spite of several attempts.

3

34

By Schultze (1202) the creatinuria in the pregnancy itself is ascribed to a temporarily increased thyreoid action. He quotes an observation of Anselm and Hoffmann, according to which the serum of pregnant women injected in animals will provoke symptoms of hyperthyreosis. Schultze himself produced creatinuria in rabbits by injecting serum from pregnant individuals, no one by injecting normal serum.

The creatine elimination during the confinement is often attri- ' buted to the involution of the uterus. But as it is not diminished by cesarean section with hysterectomia (849, 910) the cause must be another.

Mellanby (849) suggests relations to the lactation. The profound effect known to be exercised on the creatine

bodies by the sexual glands is naturally taken into consideration in this connection and it seems most probable, a t the present time, that the final explanation must be expected from this quarter. See a).

In the uterus the creatine contents are found to be elevated during pregnancy (70, 316).

In blood no significant changes have been found. Considerable work has been devoted to the examination of the mutual relations of the creatine bodies in maternal and foetal blood. It has generally been found that the values are practically identical, suggestive of a free diffusion in the placenta. See b).

Several attempts have been made upon substantiating features characteristic of pregnancy toxicoses and by some authors it is maintained that the creatine bodies under such circumstances are elevated in blood and urine, but these observations have not been generally confirmed and are of no practical significance. See c).

Fever and ofher general pathological conditions: Fever is also one of the conditions accompanied by creatine

elimination. Earlier investigators found that the excretion of creatinine

was increased during fever, a statement which has been universally a) 24, 29, 584, 672, 686, 688, 933, 934, 1128, 1317, 1386, 1445. h) 528, 548, 603, 996, 1075, 1076, 1077, 1304. c) 547, 558, 584, 585, 653, 1362.

35

accepted afterwards. When a febrile disease is leading to marasmus or passing over into convalescence the increased creatinine elimin- ation is often succeeded by a period of lowered excretion.

The occurrence of augmented creatine elimination during fever is also generally confirmed. But how the fever provokes the creatinuria still remains unexplained and the rules governing the excretion are not known.

The quantity of creatine eliminated is not always direct pro- portional to the height of the fever. On the contrary, during fever of equal height and due to the same disease the creatinuria may be high in one case and absent in another, may disappear before the fever has ceased or continue after its cessation (172, 829). In a severe attack of a disease a creatine elimination may often be found, while it is lacking in a lighter one (1425). See a).

Diathermia on the muscles (172) and artificial hyperpyresis produced by substances as dinitrophenol (726, 727, 770) have a similar effect to that of genuine fever, ordinary variations in the external temperature are found to be without influence (1439) or the creatinine increased in heat (990).

Communications concerning the blood and tissues are scarce, in the former the creatine bodies have most often been found in- creased during a fever (619, 655, 1115, 1405).

Further, creatinuria is found by wasting diseases of all kind, without regard to their origin (77).

Increase of the creatine excretion is reported to follow anoxemia (156) and cooling of animals (1012, 1014 - see also 611 and 1176), is also seen after oblockingo of the reticuloendothelial system by china-ink (932).

The relations in some diseases as in diabetes mellitus have been mentioned in other connections, the endocrine disturbances and renal retention will be considered later. After various surgical operations the urine creatinine is reported to increase (424).

Not much has been reported concerning the creatine bodies in diseases of the blood. In anemias and in levcemia the creatinine elimination is frequently found to be low. See b).

a) 172, 338, 351, 489, 575, 587, 666, 667, 726, 829, 856, 872, 929, 1059, 1092, 1169, 1221, 1224, 1241, 1425, 1426.

b) 113, 414, 463, 856, 929, 1182, 1373.

36

The vitamins are supposed to exercise some influence on the creatine bodies.

In experimental scorbut most examinators report increase of the creatine in the urine, some also of the muscle creatine, but not of the creatine in the brain (1023, 1032), only Piana (1071) finds lowered urine creatine and creatinine. Nagayama and Sat0 (962) point out the resemblance to starvation, they believe that the creatine disturbances are not due to a direct effect of the C-vitamin.

The other avitaminoses have not been so thoroughly examined. The polineuritis caused by want of the vitamin B is accompanied by increase of the creatine in brain and muscles (693, 744). Further see a).

The effect of several pharmaca and poisons on the creatine bodies has been tested, the results have partly been mentioned in other connections, the rest are a t present without any great signi- ficance for the study of the creatine metabolism. See b).

On the other hand certain effects upon the organism have been attributed to the ereatine bodies themselves. The most striking one is the power possessed by creatine of producing muscular convulsions when applied in substance upon the motoric region of the denuded brain (704, 824, 898). The effect seems to be limited to the gray matter, it is not present in peripheral nerves (824, 897).

The beats of the isolated heart is reported to be enlarged by creatine and creatinine (38, 194, 195), on the blood vessels the crea- tine bodies have no effect (32, 618, 669) or produce a slight dila- tation (147). The relation of the creatine to insuline is mentioned before. Further see c).

Local disorders - nervous and muscular diseases: As the creatine is localized principally in the muscles, important

disturbances may be expected to accompany the diseases in the latter. This does also frequently hold true.

In the first case the metabolism of the creatine bodies is inter- fered with in the pure muscular diseases. In muscular atrophies,

a) 388, 646, 901, 1021, 1024, 1070, 1438. b) 23, 26, 83, 162, 167, 175, 529, 587, 619, 649, 709, 762, 885, 932, 1007,

c) 122, 125, 196, 223, 224, 648, 773, 953, 1065, 1156, 1285, 1333, 1416. 1025, 1034, 1111, 1115, 1120, 1207, 1267, 1290, 1291, 1307, 1355, 1360.

37

without regard to their origin, the urine creatinine -the supposed measure for the muscle mass, see pag. 40 - is low (587, 1275) and the elimination of creatine is increased.

This statement comprises besides palsies the following conditions: Amputation of limbs (170), fractures (571) - both statements somewhat uncertain -, arthritis (180 1064,), inactivity atrophy (1 373).

In palsies after poliomyelitis the creatine elimination is high and the creatinine in the urine reduced. This is most evident in severe cases. (170, 419, 460, 492, 661, 721, 782.)

In myositis fibrosa creatinuria is found (112, 1268), in myositis ossificans i t has not been observed. (See 111, 460, also 458

The most striking disturbances are probably those found in trichinosis. Here enormous quantities of creatine are eliminated, while the urine creatinine is greatly reduced, both alterations being of long duration (170, 796).

Of the special neuromuscular diseases the dystrophia musculo- rum progressiva, the disease which shows the greatest muscular damage, is also that which is accompanied by the most remarkable disturbances of the creatine-creatinine metabolism, the creatine elimination being very high and that of the creatinine very low. None of these diseases has been as thoroughly studied as this one and, especially in recent years, i t has attracted great interest because of the therapeutical attempts made with administration of glycine and the changes in the creatine metabolism thereby provok- ed. These questions are of great importance for the investigation of the origin of the creatine bodies and will be treated in that section. See further a).

In the other neuromuscular diseases the damage on the meta- bolism of the creatine bodies is less prominent. In fact it may be maintained that the gross disturbances are attached to injuries befalling the peripheral neurone, and the higher up in the central nervous system the disease is located, the less the creatine bodies are affected.

Small quantities of creatine are often reported to be eliminated in diseases like amyotrophic lateral sclerosis, myotonia congenita,

a) 134, 414, 027, 780, 781, 836, 837, 856, 970, 1085, 1100, 1259, 1204.

. and 537.)

38

tabes dorsalis, hemiplegia and multiple sclerosis, t o this group may also be reckoned injuries on the spinal cord.

On the creatinine excretion the opinions are various, both increased, unaltered and lowered elimination have been reported. Thus in tabes dorsalis augmented creatinine in the urine has been observed when the patient has been up and moving about and reduced when he has stayed in bed (1241). See a).

Concerning the myasthenia gravis there are some disagreements, by some investigators creatinuria has been found, by others not - see b).

In some cerebral diseases augmented creatine elimination has been reported, as in postencephalitic parkinsonism, but this has been denied by other authors.

The most debated question in connection with the diseases in the upper parts of the central nervous system is, however, another: It has frequently been maintained that in the hypertonic conditions so common in these disorders the creatinine in the urineis increased. But the opposite view has also numerous supporters. See pag. 26.

In mental diseases no significant effect upon the creatine bodies have. been pointed out.

The observation of Weinberg, that the creatinine in the urine is augmented by ))preoccupied mind)) is probably due t o a technical error (1402).

Several examinations of the creatine and the creatinine in blood in the above diseases have been reported, but the communica- tions' are of little interest, they are here grouped with those on the urine. See c). (As more diseases are often treated together, the division cannot be complete.)

Most important are the observations made on excised muscular material, especially from dystrophia musculorum progressiva, myasthenia gravis and muscles paralyzed by division of the nerves. In the first and in the last case the creatine contents of the muscles are reduced, in myasthenia gravis they are normal. These facts are of great interest compared to the urine examinations under these conditions. See pag. 23 and d).

a) 170, 189 754, 856, 975, 1133, 1148, 1452,.. b) 118, 305, 837, 877, 1105, 1414. c) 247, 319. 364, 520, 564, 569, 570, 1009, 1093, 1170, 1376, 1388, 1435, d) 36, 201, 245, 696, 982, 10152, 1115, 1120, 1206, 1282.

39

In this connection may be remembered that creatinuria is provoked by treatment of the muscles by diathermia (172) and perhaps also that the same result is attained by cooling of animals in a cold bath (1012, 1014).

With some hesitation I mention in this section the finding of Bohn (1 13, 114) that epale hypertoniai) is constantly accompanied by an elevated creatine elimination, which is not seen in wed hypertoniai), Bohn therefore assumes that a muscular affection is present in the former case, not in the latter.

Perhaps the communication of .Kindler (657) also ought to be considered here, reporting the occurrence of creatinuria in cases of cardial incompensation, ascribed to anoxemia. This assumption is supported by the work of Brunquist, Schneller and Loewenhart, who were experimenting with reduced oxygen contents in the respiratory air, resulting in creatinuria (156).

Further belong to this section the experiments of Hermann, Decherd and collaborators (551, 552), producing an artificial occlu- sion of the coronary vessels, accompanied by reduced creatine con- tents in the injured parts of the heart, and augmented creatine in blood and urine.

Mutual relations of creatine and creatinine, and the effect of admi- nistration of the creatine bodies perorally or parenterally:

The near chemical relationship between the two creatine bodies has already from the time of their discovery led to the assumption that they are also closely connected physiologically, a view which has been retained later by most investigators, in fact as a postulate. For it has turned out that the proof for the existence of this connection is difficult to furnish and has perhaps not been furnished in a wholIy convincing way till this day.

So great are the difficulties, that several authors, impressed by the negative results, have concluded that there does not exist any relation a t all between the two substances.

It is generally believed, however, that creatinine is formed from creatine - and not converse, with one exception (634).

First of all the observations must be mentioned made from a pure chemical point of view on the transition of creatine t o crea- tinine and vice-versa.

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In an aqueus solution of creatine, creatinine or both, one of the substances is gradually transformed into the other. This process goes on until an equilibrium has been reached, the final result being identical whether i t is started from a creatine- or from a creatinine solution.

In the presence of hydrochloric acid the conversion goes only from creatine to creatinine, the difference probably being due t o formation of salts of the more basic creatinine with hydrochloric acid.

The speed of the conversion is affected by pH and temperature, the final result not. See a).

I t lay close a t hand to believe that the daily output of crea- tinine originated from the muscular creatine under the influence of hydrogen ion concentration and temperature in the muscles and in some publications i t has been attempted to make this probable (514, 516, 947).

Other authors think that the action of certain enzymes must be supposed and maintain having detected such, most often asso- ciated with enzymes having the ability of decomposing creatine and creatinine. Most of these statements are based upon experiments on autolysis and perfusion.

Mellanby (848) and others reject the theory of the action of enzymes, Hammet (525) thinks that the conversion creatine- creatinine is ))biocatalyzed)) by the milieu of the living muscle.

In favour of the assumed origin of the urine creatinine from the creatine of the muscles i t has been reported that the former is reduced when the latter is low (286) and that there is a constant relationship between the two values when various animal species with different muscle creatine are compared (938), the last-mention- ed statement has, however, been rejected by other authors (213, 1315). See b).

Most attempts a t approaching this problem have been made by administration of creatine and creatinine per 0s or by subcutane or intravenous injection.

a) 66, 182, 191, 323, 324, 509. b) 15, 16, 29, 70, 76, 90, 449, 465, 466, 523, 524, 527, 573, 579, 587, 656,

736,1002, 1150, 1154,1174, 1175, 1236,1264, 1378.

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These experiments have revealed some peculiar features in the metabolism of the creatine bodies.

When creatine is administered in one of the ways mentioned, smaller quantities will produce no changes in the creatine contents of the urine. After somewhat larger doses - a limit of 1-2 g has often been mentioned (1079, 1451), - the creatine elimination in the urine begins to rise.

But as a rule the whole quantity of creatine administered will not appear, on the contrary only ca. 25-50 yo is retrieved in healthy subjects, less in men than in women. In children a some- what greater elimination has been found, up to 75 %. Reich (1100) thinks that the less of the creatine administered is eliminated, the better the musculature of the individual is developed. See a).

This creatinuria due to administration of creatine, is designated as ))exogenous creatinuriao in contradistinction to the ))endogenous)) or ospontanouso creatinuria caused by one or the other of the circumstances mentioned, physiological or pathological.

The quantity of administered creatine which is retained by the organism, is a measure for its so-called xreatine tolerance)).

The exogenous creatinuria respectively the creatine tolerance are regarded with special interest as possible means for testing the state of the creatine metaboIism.

In the first place, of.course, the objects for testing are indi- viduals showing some kind of spontanous creatinuria. It is a common statement that the tolerance for exogenous creatine is reduced in such conditions and proportional to the quantities of endogenous creatine eliminated.

Thus Wolff (1429) finds that low creatine tolerance and spontanous creatinuria are independent of one another, both may occur alone. He still attributes some importance to the tolerance test.

But recently Espersen and Thomsen (349) publish a crushing criticism of the creatine toIerance test. They point out the great difficulties in judging the amount of exogenous creatine eliminated on account of the considerable irregularities of the spontanous creatinuria, and they think that these difficulties have caused great errors. In neuromuscular diseases, especially in dystrophia muscu-

This assertion, however, has not escaped objections.

a) 90, 151, 152, 209, 354, 687, 798, 845, 1063, 1087, 1113, 1265.

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lorum, they find no relations between endogenous and exogenous creatinuria.

The tolerance test has most often been applied to neuromuscular and endocrine disorders and will be returned to in connection with the latter.

The effect of creatine administered upon the creatinine excreted, intended to give the clue to the question of the relation between the two creatine bodies, is not easy to interpret. At any rate it is not very conspicuous.

The earliest authors supposed that creatine ingested increased the urine creatinine and thus underwent a partial conversion in the body (846, 929, 1379). But Folin (397) rejected this and declared that no conversion occurred and that the two creatine bodies were quite independent. He was supported by Wolf, Shaffer and others and for some time his view was prevalent. (164, 405, 713, 1428.)

Some authors, however, found a slight increase of urine crea- tinine, especially after repeated ingestion of creatine. Assuming that the conversion was a very slow process, Rose, Ellis and Helming (1144) and Benedict and Osterberg (84) tried prolonged administra- tion of creatine and after some time found a considerable increase of the creatinine. But Hahn and Fasold (512) failed to reproduce the experiment. See a).

It must, therefore, be concluded, that the conversion of creatine to creatinine in the organism has not yet been indisputably proved, even if it is probable. In the same way it may be said that the proof for the origin of the creatinine of the urine from the creatine of the muscles has not been delivered. But this process is openly or tacitly assumed by most authors.

By administration of creatine the creatine contents of blood, liver and muscles are raised, but the increase is small and, even by continued supplying with creatine i t has been reported to be transitory. Especially in the muscles a maturation level, is supposed to exist. See a) and b).

In comparing the creatine administered with the creatine stored in the organism and excreted as creatine and as creatinine - con- version assumed - all investigators report the peculiar result that

a) 207, 587, 767, 944, 1061, 1127, 1141, 1398. b) 103, 208, 211, 216, 218, 219, 530, 539.

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a considerable quantity of creatine cannot be accounted for, it is dost)), and where is still quite unknown, even the nitrogen eli- mination has not been found to be influenced, nor have other disintegration products been demonstrated. See a).

It appears therefore that parts of the creatine metabolism are as yet wholly unknown and may differ completely from those of which we possess some knowledge.

Of creatinine administered about 80 yo is eliminated in the urine.

Twort and Mellanby (1352) and others fear that creatine given per 0s may suffer losses in the gut, as they report the existence of a creatine-destroying microbe here. But the amount of creatine lost is practically the same by subcutane or intravenous injection (117) and the method of administration is considered to be un- important. See b).

In the later years the question of the mutual relations of the creatine bodies is remarkably neglected.

The origin of creatine: Of all the questions concerning the creatine metabolism, this

is undoubtedly the most extensively discussed. A vast quantity of experimental work has been displayed, but the result has been an utterly indecisive multitude of contradictions.

The experiments comprise administration of the supposed mother substances of the creatine to man or animals per 0s or by injection, the effect produced on the creatine bodies in the urine being watched. Sometimes alterations of the creatine in the muscles are also sought for.

Further it has been attempted to demonstrate formation of creatine by perfusion of surviving organs after addition of various substances to the perfusion fluid.

Moreover the same process has been tried in autolyzing organ- ,Breio and even in living, isolated tissues (127).

I t may be stated a t once that none of the experiments have resulted in an ample transformation into creatine, thus absolutely convincing proofs have not been provided. A great many investig- ators deny any creatine formation in these experiments or admit

a) 218, 586, 665, 941. b) 207, 767, 944, 1087.

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that i t is not distinctly shown. But several authors maintain that one or another of the substances tested provokes an increase of creatine.

The number of the substances suggested as sources of creatine formation is exceedingly great and their nature widely different.

As the most important ones may be mentioned the guanidine derivates, pointed out by their near chemical relationship to the creatine, and first among them the important and commonly occurr- ing arginine.

The formulae for the transformation of arginine to creatine are easily put down (1) and the reciprocal occurrence of arginine and creatine in invertebrates and vertebrates points towards a relation, but the unmistakable proof is lacking in spite of innumerable and ingenious experiments. See a).

The hypothesis is forwarded that the arginine may be broken down in two different ways, one of them leading to creatine, the other by the action of arginase to ornithine and urea and bet- ween these ways a competition is supposed to exist. But attempts a t protection of the arginine from the action of the arginase by various substitutions have been without results. See b).

The glycine stands next to the guanidine derivates in importance, and that for practical more than theoretical reasons. Its position is owing to its employment as a therapeuticum in neuromuscular diseases, especially in dystrophia musculorum progressiva, so extensively tried during the later years.

In dystrophia musculorum treated with glycine practically all authors find an increase of the creatine elimination. Many of them further report that this increase is transitory even by continued administration of glycine and is followed by a decrease which is sometimes found t o proceed to values lower than the initial level of creatine elimination.

According to some investigators this decrease is accompanied by an increase of the creatinine elimination and a corresponding improvement of the patient. But while the first assertion is gener-

a) 2, 3, 4, 6, 7, 53, 56, 86, 161, 260, 326, 365, 469, 493, 526, 580, 605, 608, 615, 625, 628, 697, 726, 855, 946, 1055, 1056, 1083, 1109, 1140, 1214, 1227, 1244, 1288, 1289, 1326, 1329, 1330, 1424.

b) 150, 494, 1319, 1321, 1322, 1323, 1324.

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ally accepted, the others are objects of increasing diversities of opinion and seem to be denied by most of the recent authors.

Besides in the dystrophia musculorum the effect of glycine medication is also reported to occur in other neuromuscular diseases, particulary in myasthenia gravis, though not so pronounced, and it is found in thyreotoxicosis and even in normal individuals (37, 157, 778, 1052, 1233, 1354). See a).

The possible ways for the formation of creatine from glycine are given by Brand and Harris (133).

Of other supposed mother substances for the creatine may be mentioned the choline and the betaine (5, 8, 53, 581, 1116, 1117, 1226, 1229) and the glycocyamine (52, 266, 310, 459, 625, 1035).

But further are suggested practically all other amino acids - certain investigators (60, 61, 63) report creatine formation from a bewildering multitude of these substances, of which most are tested by others with negative results. See also b).

All this seems not to be very convincing, the alterations reported are relatively small and the objections numerous. The question therefore ought to be regarded with extreme caution. As far as I am aware all observations are based upon colorimetric estimations and no attempt made to verify these by isolation of the creatine bodies presumably formed. It must be remembered that the reaction of Jaffe is by no means specific and that e.g. the guanidine derivates are supposed to interfere. According to my view the possibility cannot be excluded that the gross administration of biochemically important substances commonly employed in these experiments, may not generate excretory products other than creatine bodies, which are able to influence the colorimetric readings.

Many authors hold that all attempts at demonstrating exo- genous creatine formation by administration of the mother substance in excess must be futile as the creatine, having a distinct function in the organism, is only formed in the quantities wanted for the fulfilment of this. This view seems to be excellently supported by the recent discoveries of the phosphocreatine.

a) 18, 19, 20, 25, 64, 65, 67, 108, 119, 120, 133, 135, 136, 137, 157, 212, 265, 491, 492,535, 546, 563, 660, 683, 684, 740, 778, 876, 878, 879,880,1066, 1100, 1102, 1191, 1284, 1320, 1325, 1333, 1348, 1349.

b) 62, 100, 103, 104, 117, 227, 254, 276, 459, 504, 534, 553, 670, 713, 727, 774, 883, 985, 1067, 1205, 1217, 1271, 1272, 1361, 1449, 1454.

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That creatine may really be synthetized in the organism is shown, for one, by the starvation experiments mentioned before - p. 27.

Phosphocreat ine: The epoch-making discovery of this compound was preceded

by several observations, to whose significance i t gave the clue. In a very amusing article Hill (560) has enumerated the previous occasions a t which the phosphocreatine was onearly discovered)). Hartree and Hill found the udelayed contraction heat)) in 1920, in 1923 Hoppe-Seyler demonstrated more rapid diffusion of phos- phates from fatigued than from rested muscles, for creatine the same was reported by Urano (1364) and by Tiegs (1337). In 1924 Embden demonstrated the delayed lactic acid production in muscular contractions.

Late in the year 1926 came the report by Eggleton and Eggleton of the existence of a labile phosphoric acid compound in the muscles (332) and shortly afterwards and independent thereof appeared the communication of Fiske and Subbarow (384) of the same result.

The latter, however, compensated for holding the second place by recognizing that the other constituent of the labile compound was the creatine, while Eggleton and Eggleton meant that it was some carbohydrate (332, 333).

But these authors won back their position by realizing that the phosphocreatine (they called it ophosphagene))) was of importance for the contraction of the muscles, being split during this and resynthetized afterwards in the presence of oxygene. Fiske and Subbarow, on their side, regarded the phosphocreatine only as one of the buffer substances of the muscle, employed in neutralizing the lactic acid formed during contraction.

The discovery of the phosphocreatine a t once started a vivid investigation, a t first mainly confined to confirmations of the initial results. See a).

But soon progress was made: Phosphocreatine was found also in heart, unstriated muscles and nerves, possibly in liver and blood

a) 48, 123, 130, 178, 268, 273, 307, 315, 318, 331, 334, 335, 336, 353, 370, 371, 375, 383, 385, 464, 716, 799, 801, 802, 804, 805, 807, 811, 858, 8G8, 869, 887, 999, 1017, 1019, 1026, 1110, 1238, 1300.

47

-see a). Its occurrence in the animal kingdom is mentioned before - pag. 13.

Already in 1927 Meyerhof and Lohmann (863,864) demonstrated the great heat production by hydrolytic splitting of the phospho- creatine, ca. 120 cal. per g H,PO,, and shortly afterwards Nach- mansohn (954,955) demonstrated that some of the phosphocreatine was resynthetized anaerobically.

Thereby the investigators came a t a loss. As the splitting of the phosphocreatine is a strongly exotherm process, the resynthesis must be to the same degree endotherm and in anaerobic conditions no process could then be imagined, which was able to deliver the necessary energy.

Therefore the theory was set forth that in the muscular con- traction the phosphocreatine was not really split, but only made *instabile)), which for the methods employed had the appearance of a splitting.

There was also some incertainty concerning the part played by the phosphocreatine and diverging views were put forward, some of them being contended by certain authors up to now. Thus Nachmansohn a t first regarded the phosphocreatine as con- nected with the irritability of the muscles, according to Martino i t was of special importance to the neuromuscular connection. Mackler, Olmstead and Simpson and Sacks and Sacks have main- tained the original view of Fiske and Subbarow, that the phospho- creatine is a buffer substance. It seems, by the way, that it really possesses the power of neutralizing some lactic acid when splitting a t the p H of the muscles (861). See b).

But i t was from the investigations of Lundsgaard that progress now came. By poisoning of surviving muscles by monoiodacetic acid (CH,ICOOH) the lactic acid production during contraction is prevented and i t is possible to study the phosphocreatine as uninfluenced by the lactic acid.

a) Kervous system: 374, 450, 451, 452, 453, 562, 576, 651, 819, 1294. Heart and unstriated muscles: 235, 236, 374, 616, 785, 803, 806, 813,

Blood: 10, 179, 230, 582, 583, 652, 889, 965, 1295. 896, 922, 1005, 1080, 1082, 1173, 1216, 1239, 1240,1382,1399, 1447, 1448.

b) 176, 177, 775, 776, 800, 808, 809, 810, 812, 956, 957, 989, 1158, 1159, 1160, 1161.

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In living animals poisoning with monoiodacetic acid rapidly produces a violent rigor with ensuing death.

Lundsgaard (756, 757, 758, 759) demonstrated that muscles poisoned in this way are able to contract anaerobically for a short time, then ending in rigor. During these contractions' the phos- phocreatine is split, the rigor coinciding with the complete exhaus- tion of the phosphocreatine depots. Between the work thereby executed by the muscle and the heat produced by the splitting of the phosphocreatine he found a very fair accordance.

In muscles not poisoned the relation between the phosphocrea- tine splitting and the work yielded is partly masked, also under anaerobic conditions, by an immediately occurring resynthesis of the phosphocreatine. This is furnished with energy from the hydro- lytic breakdown of glycogen through various intermediary products into lactic acid, a process starting after the beginning of the phos- phocreatine splitting and continuing for some time after the accom- plishment of the contraction, thus explaining most of the ))delayed contraction heat)).

Under aerobic conditions the matters are still more complicated on account of the partial resynthesis of glycogen from lactic acid, on the expence of the oxydative breakdown of a fraction of the carbohydrate splitting products into carbonic acid and water.

Thus, the energy for the contraction is first provided by a series of rapid, anaerobic splitting processes, each of which is immediately restored by e