6I :6I2.396.I4

Transcription

1 6I :6I2.396.I4 THE METABOLISM OF LACTOSE. Part I. The occurrence of lactose in urine. BY L. B. WINT E R (Grocers' Company Research Scholar).. (From the Sherrington School of Physiology, St Thomas's Hospital. THE development of the mammary gland during pregnancy presents many problems for study, and one of the most interesting is that which is concerned with sugar metabolism. Lactose is the chief, if not the only, sugar present in the milk of all mammals, and this is its sole natural source. Why the young mammal should be supplied with carbohydrate in this form is uinknown; the livers of foetal and young animals contain glycogen, and until evidence to the contrary is presented it may be assumed that this breaks down to supply glucose for energy as in the adult. That galactose is required for synthesis of cerebrosides is unlikely; brain tissue is formed in the foetus, the necessary galactose being most probably formed by molecular rearrangement from glucose supplied by the maternal blood; there is no reason to suppose that this process cannot continue after birth. The mechanism by which lactose is secreted in the milk has not yet been solved; that the disaccharide is formed in the mammary gland from glucose is suggested by Porcher's experiments [1904]. Porcher considered that if lactose was formed elsewhere than in the mammary gland, removal of the gland would lead to excretion of lactose in the urine, which could be identified by formation of the phenyl-osazone. The breasts of goats were amputated, and the only osazone which could be isolated from the urine subsequent to parturition was that of glucose. No direct evidence as to the formation of lactose by the mammary gland is available. All attempts to show the presence of an enzyme in the gland which can convert glucose or glucose and galactose into lactose have been unsuccessful; the most recent are those of Svanberg [1930]. Whatever the mechanism may be by which lactose is formed by the maternal tissues, the adjustment of the factors involved is often by no means perfect. It has been known for many years that reducing sugar may often be found in the urine of healthy lactating women, and less frequently during pregnancy. The nature of the sugar PH. LXXI. 23

2 342 L. B. WINTER. excreted after parturition was the subject of controversy, until Hofmeister [1877] isolated the sugar and found that it agreed in all its properties with those of lactose. Lemaire [1895], using the benzoyl chloride method followed by condensation of the sugar with phenylhydrazine, stated that lactosazone could be readily identified from urines during the lactation period, but that previous to parturition glucose was the only sugar which could be detected. Recently Watkins [1928] has made a detailed study of the excretion of reducing substances by women during pregnancy and lactation. Sugar was estimated colorimetrically, following fermentation of the urine by yeast. An increased amount of non-fermentable "sugar" was found in the urines towards the end of pregnancy, rising sharply just before delivery. After parturition there was a fall for some days, followed by a sudden and often very large increase about the fifth day of the puerperium. Lactose is only one of the non-fermentable sugars which might be excreted at the time when the mammary gland is beginning to adjust itself to the excretion of lactose in the milk. Watkins considers that lactose is secreted during pregnancy and after parturition, and this view is shared by Harrison [1930]. The metabolism of lactose by non-pregnant women was also studied by Watkins, and it was found that the tolerance dose for the ingested sugar was normally about 10 g., agreeing with the figure obtained for normal men by Folin and Berglund [1922], but that during menstruation it was increased to 20 or 30 g. Lactose is a disaccharide, and either component sugar, or the unchanged biose, might be excreted following ingestion, yet in all the investigations carried out in recent years the decision has been reached by estimation of "sugar" in urine without any evidence as to what substance was being estimated. The present work was begun with the object of approaching lactose metabolism more from the qualitative standpoint. Urine sugar after parturition. In a series of women after normal labour the urine was examined for sugar each morning during the puerperium. The test consisted in boiling an equal volume of urine and Fehling's solution for seconds and allowing to stand; if no noticeable reduction occurred during the next minute the test was considered negative. Fehling's test is comparatively insensitive, and is falling into disuse, but it was chosen deliberately for the reason that by it normal and abnormal sugar excretion could be at once differentiated. Many healthy individuals pass urine which gives a slight reduction of Fehling's solution; any urine which

3 LACTOSE IN URINE. 343 does not must certainly contain sugar within limits which are normal. The nature of the normal urine "sugar" is uncertain and probably varies much according to the type of food taken; apart from the question of lactose excretion this problem is one for special and detailed study; it seemed undesirable at the present stage to consider any sugar excretion which was normal in amount. The statement that the excretion of reducing sugar after parturition is common was confirmed. Of twenty-nine cases examined, all were sugar-free on the day after delivery, yet sixteen gave a positive test for sugar later in the puerperium. The sugar appeared usuallyonthethirdday, andin some cases the reduction was very marked, simulating that in cases of diabetes. No reason could be assigned clinically for the appearance of sugar in the urine; there was no apparent difference in type, e.g. age, parity, or quantity of milk secreted, between those cases which excreted sugar and those which did not. The one significant fact was the coincidence of the sugar in the urine with the coming in of the milk, suggesting that in many women, normal in other respects, the adjustment of carbohydrate metabolism at this time was temporarily upset. This derangement might be of two types, a reabsorption into the blood of sugar formed in the mammary gland, or sugar carried to the gland from, for example, the liver, in amount greater than the gland was able to use. The latter possibility must be considered, although it is known [Rowley, 1922] that during lactation the blood sugar is not increased significantly in amount; whether it differs qualitatively from normal is being investigated. As adjustment becomes more perfect the sugar in the urine decreases, and most cases were sugar free on leaving hospital 10 days after delivery. It was possible that the sugar excretion at the begin ing of lactation might throw some light on the immediate precursors from which lactose is formed, and with this end in view the nature of the sugar excreted has been investigated as completely as possible. Lactose phenyl-osazone. The formation of phenyl-osazones is often used as a means of identification of sugar in urine, and this method was first tried. It is difficult to prepare samples of osazones, especially from disaccharides, which show correct analyses and sharp melting points, and the latter do not differ much for the derivatives of different sugars. The optical properties are widely different, and the determination of this constant affords more useful evidence. As a preliminary, some earlier observations on the phenyl-osazone of lactose were confirmed. This compound, described by Neuberg [1899] as inactive in pyridine-alcohol, shows an appreciable 23-2

4 344 L. B. WINTER. positive rotation in this solvent, with mutarotation. Owing to the deep colour of the fluid special precautions must be taken when making the observations [Winter, 1925]. 2 g. lactose hydrate, 2-8.g. phenylhydrazine hydrochloride, and 2*5 g. sodium acetate were dissolved in 50 c.c. water and heated on the boiling water bath for 30 minutes. After cooling, the crystals were separated, washed with cold water, recrystallized from hot aqueous pyridine, and dried over sulphuric acid in a vacuum. Heated rapidly in a capillary tube the substance sintered at 194 and melted at (uncorr.). 0*1026 g. in 4*588 g. pyridinealcohol (d ). c = Time (hours) a (a] Micro-analysis (by Dr Ing. A. Schoeller, Berlin): C 54-25, 54-19; H 6*34, 6*24; N 10*60, 10*57. C12H4O9 (N.NH.C6H5)2 requires C 55-38, H 6-15, N 10*76. Samples of urine which gave a vigorous reduction of Fehling's solution, obtained from cases 4 or 5 days after parturition, were placed in the boiling water bath with excess of phenyl-hydrazine and acetic acid. The phenyl-hydrazine had been just previously distilled at low pressure and was almost colourless. After heating for 30 minutes the solution was allowed to cool slowly. The flocculent precipitate was centrifuged, washed with cold water, and extracted with hot water in which it was partly soluble. Yellow crystals were deposited on cooling; after one further crystallization the deposit was observed microscopically. The crystalline form varied in different experiments, but the most common was star-shaped groups of broad needles. No crystals typical of lactosazone were ever observed. For identification by melting point and rotatory power it was necessary to work on a larger scale. Two methods were used; the first was essentially that used by Needham [1924] for the extraction of inositol from urine c.c. urine were made into a dry mass with plaster of Paris and the solid was extracted with 85 p.c. alcohol. The alcohol was evaporated under low pressure, and the aqueous residue was used for preparation of osazones. In the second method an excess of neutral lead acetate was added to the urine, the precipitate was filtered off, and after removal of lead as sulphide the solution was concentrated in vacuo to a small volume. From most urines it was 1 All rotations were observed in a 1 dm. tube. The polarimeter wais a three field instrament, and a half shadow angle of 1 was used. In this paper figures for the specific rotation of lactose are calculated for the hydrate.

5 LACTOSE IN URINE. possible to separate by either method an osazone insoluble in hot water, with the crystalline form, melting point, and rotatory power of glucosazone, but in no case was the fraction soluble in hot water identifiable with the osazone of lactose. Even after four or five crystallizations the star-shaped type of crystal persisted; the melting point was very variable, and optical activity was absent or markedly negative. This line of attack was abandoned, and it was decided to separate whatever sugars might be present in a pure form and to determine their physical constants. Separation of lactose added to normal urine. The method used has recently been applied to the identification of the blood sugar, and described in that connection [Winter, 1930]. Further description is unnecessary here except to note that no concentration of urine is carried out; an excess of lead acetate is added, the mixture filtered, and the filtrate is made alkaline with ammonia. The following experiment shows that when lactose is added to urine much can be recovered sufficiently pure for identification by melting point and rotatory power. 1-0 g. lactose hydrate added to 200 c.c. urine from a healthy man. The lead precipitate weighed 13-5 g. The sugar was recrystallized from dilute alcohol, yield 0-5 g. By working up the mother liquor a further 0 18 g. was obtained. The mixed sample gave the following figures. M.P g. in 5-01 g. water. c = Initial Final a = [a] C 40-25, 40-42; H 6-51, C12H -O11.H20 requires C 4000, H With care it is possible to make the separation of the sugar roughly quantitative, but with a large number of experiments it saves much time if only the first crop of crystals is weighed. On this basis a 50 p.c. yield should be obtained, and this figure was used for comparison when the lactose was extracted from the urine of lactating women. Examination of the sugar excreted after delivery. Estimations of the reducing value of the fresh specimens of urine were carried out by the Wood-Ost method, the permanganate being standardized in the usual way, and also by checking against known amounts of pure glucose and lactose in water. The sugar was then isolated from the urine and the yield compared with that deduced by titration.

6 346 L. B. WINTER. M. W. Age 30. Multipara. 72 c.c. urine (drawn off by catheter) containing 1*62 g. sugar calculated as lactose hydrate g. sugar isolated which gave the following figures. M.P g. in 9-98g. water. c = 2-0. Initial Final a= F21 [a]1= C 40-26, 39-93; H 6-48, 6'66. 0*25 g. of the substance was dissolved in 6 c.c. water with the addition of 0*3 g. sodium acetate and 0-35 g. phenyl-hydrazine hydrochloride, and heated in the water bath for 30 minutes. The precipitate which deposited on cooling was examined microscopically and found to be typical of lactosazone. The product was recrystallized from hot dilute pyridine. M.P with sintering at g. in 1F754 g. pyridine-alcohol (d ). c = 1 2. Initial Final a= [a] C 55 40, 55-33; H 6-34, 6-09; N 10-80, The yield of lactose in this experiment is 33 p.c. of that "sugar" content found by titration. But the estimation of reducing power was made in urine, and substances other than sugar reduce the copper solution, therefore the result was too high for this reason; it was found also that lactose added to normal urine whose reducing power was known was estimated too high after the correction for the normal urinary constituents had been made. The increase is fairly constant with samples of urine of different reducing power, and was found to be 10 p.c. The explanation is not clear, but the following are representative figures. " Sugar " (g.) Lactose (g.) Lactose (g.) in 100 c.c. added to Error urine 100 c.c. Found Corrected (p.c.) * Since there is no reason to suppose that the normal urinary reducing substances are diminished in amount during pregnancy and lactation, it seems fair to assume that the apparent lactose content is 20 p.c. too high at a conservative estimate, and that the yield of sugar obtained from these urines is not significantly low. Nevertheless the mother liquor has been worked up to detect other sugars which might be present, viz. glucose, galactose and cellobiose. Any of these sugars might be

7 LACTOSE IN URINE. concerned in the formation of lactose, and be excreted in the urine at the time when the mammary gland was beginning to secrete milk, before perfect adjustment had taken place. Supposing one of these sugars to be present, they should be detected after removal of the greater part of the lactose, as follows. Glucose and galactose, by a significant raising of the specific rotatory power of the mixed crystals, and lowering of the melting point; cellobiose (which crystallizes as the,8 compound at room temperature), by lowering the specific rotation and masking the course of mutarotation, and by raising the melting point. The mother liquor from the urine of M. W. was subjected to fractional crystallization from dilute alcohol with the following result. Yield, Fraction (mg.) M.P. Initial Final A B a D These figures do not suggest that there was present in the urine any significant quantity of sugar other than lactose. E. H. Age 24. Primipara. Urine taken 4 days after parturition. 138 c.c. containing 2-65 g. sugar calculated as lactose hydrate. 1P02 g. sugar isolated. M.P g. in 2-01 g. water. c = 2-5. Initial Final a= [a] = C 40-15, 40-01; H 6-55, Phenyl-osazone: M.P [a]461 = +33 (in pyridine-alcohol). Fractionation of the mother liquor. [a] W1l Yield, Fraction (mg.) M.P. Initial Final A B Lactose being a disaccharide, the reducing power is approximately doubled after boiling with acid. In every case examined in which sugar has been excreted during the puerperium, and the reducing power of the urine increased to this extent under standard conditions of heating, when the sugar has been isolated it has been found to be lactose and no other sugar has yet been detected. The conditions found most suitable for complete hydrolysis without appreciable destruction of sugar are an acid content of half normal HCl, and heating under reflux at 1000 for 13 hours.

8 348 L. B. WINTER. The sugar excreted before parturition. Twenty-seven cases were examined immediately before parturition. Only two passed urine which reduced Fehling's solution: in both of these cases heating the urine with acid caused no increase in reducing power, indicating that a mono-saccharide was present. The sugar proved to be glucose. The following are the experimental results. A. C. Age 24. Primipara. 87 c.c. urine drawn off by catheter 3 hours before delivery. 5 c.c. urine were diluted with an equal amount of N HCI. 4 c.c. required 2*8 c.c. permanganate before and after heating. 81 c.c. urine, containing 0*33 g. sugar calculated as glucose, were worked up for separation of the sugar. The final syrup crystallized after stirring, and in the course of a day became a hard dry mass. This was broken up, rapidly washed with alcohol, and dried over sulphuric acid. Yield 95 mg. 50 mg. were dissolved in 4 c.c. water with the addition of the calculated amounts of sodium acetate and phenyl-hydrazine hydrochloride, and the mixture was heated for 30 minutes on the boiling water bath. After cooling, the solid was recrystallized from hot dilute alcohol. Yield 20 mg. M.P , unchanged when intimately mixed with a known sample of glucose phenyl-osazone. 15 mg. were dissolved in 2-01 c.c. of pyridinealcohol (d ). c = 0*74. Initial Final a = (a]s4l= The remainder of the sugar was recrystallized from dry methyl alcohol for analysis. M.P *2 mg. were dissolved in 1*704 g. water. c= Time (hours) a * [a]646l C 39*87, 39'91; H 6-49, 6*47. M. C. Age 40. Primipara. Urine obtained 12 hours before parturition. 5 c.c. urine were diluted with 5 c.c. N HCI, and 4 c.c. required 6-2 c.c. permanganate before, and 6*4 c.c. after hydrolysis. 220 c.c. urine, containing 2 g. sugar calculated as glucose, were precipitated with lead acetate and the sugar was extracted. Yield 0 70 g. M.P a]= falling to C 39x87, 39-99; H 6*54, Phenyl-osazone: M.P [a]561=-78 falling to - 40.

9 LACTOSE IN URINE. In both of these cases glucose was replaced by lactose in the urine after parturition. The following are the sugar estimations carried out daily on M. C. A sample was obtained at 6.0 a.m. each morning, but 24-hour specimens were not collected, and no estimate of the total daily sugar excretion was made as the point of interest was the cessation of glucose excretion after parturition and the appearance of lactose. "Sugar" Before hydrolysis (p.c.) After hydrolysis (p.c.) 349 May 10th th * 12th 0* th 0* th th 0' * Delivered at 6.0 p.m. Utilization of lactose. The excretion of lactose in the urine at the time when the milk is coming in is often considerable in amount, a sugar content of 2 p.c. being not uncommon. It is possible that the sugar excretion is due to reabsorption into the blood of lactose formed by the cells of the mammary gland, owing to temporary blocking of some small ducts, for no significant alteration in the lactose excretion took place when the milk was drawn off completely from the breasts at frequent intervals; on the other hand, when the breasts were strapped up and exit of milk prevented, as in cases of still-birth, there was no sugar excretion above that found in women who were feeding their babies; evidently the mechanism stops rapidly under the lack of the stimulus of suckling. It is possible that the mammary gland during development in pregnancy produces lactose which is reabsorbed and excreted, but no identifiable amount passes into the urine at this time. If however, as suggested by Watkins, lactose is a utilizable sugar, and also is excreted before parturition, then the amount of lactose in the urine is not the total which is produced, but much more must be formed by the gland and utilized by the maternal tissues, only the excess appearing in the urine. Such a view implies a considerable misdirection of effort on the part of the gland. Formerly it was considered that lactose, when introduced into the circulation before being split into its constituent sugars, was quite unassimilable. Watkins argues that after a meal of lactose, unsplit sugar passes the gut wall, but in the case of small doses it does not appear in the urine because it is utilized. The results obtained by the present writer show that when lactose is given by the mouth very little passes into the

10 350 L. B. WINTER. circulation even after 50 g. doses. With smaller amounts, if any is absorbed unhydrolysed, it is more probable that it escapes detection in the urine owing to slow excretion and imperfect methods of analysis, than that it is utilized by the tissues. I have found no change in the lactose excretion of parturient women after giving insulin. Though the blood glucose is the only sugar known to be influenced, nevertheless the diminution of this sugar following insulin injection might lead to the circulating lactose being called upon, were it oxidizable. All experiments on these lines gave negative results. Watkins gave 10, 20 or 30 g. doses of lactose by the mouth to women in order to determine what amount would lead to an increased excretion of non-fermentable reducing substance. No distinction was made between lactose or galactose. It was found that for women during the inter-menstrual period the tolerance dose was 10 g., but that during menstruation or pregnancy, but not during lactation, g. could be taken without excreting extra sugar. In the following experiments 50 g. of lactose were taken, irrespective of body weight; the conditions were similar to those used by Watkins, the sugar being given about 3j hours after a light breakfast, but as the experiments were often long continued, the subjects (medical students) were not required to abstain from food longer than 7 hours. Two sugar estimations were carried out on each sample of urine, before and after boiling with acid; since by the method used no increase in reducing power is detected in normal urine after acid hydrolysis, it was easy to discover at what time after ingestion of lactose any unsplit sugar began to be exereted. In the experiments of Folin and Berglund [1922] this was not possible, as the urines showed an increase in reducing power on heating with acid before ingestion of the sugar, and throughout the experiment. The following are representative experiments and show that as a result of taking 50 g. lactose the amount of unchanged sugar excreted in the urine must be very small. To suppose that the figures are reliable for the absolute amount of lactose excreted would be incorrect, bearing in mind the presence of other reducing substances, and the high values obtained for lactose in urine. The purpose is to show that the lactose excretion after taking the sugar by the mouth is so small and spread out over so long a time that no method not specific for this sugar can give the actual values, and that different individuals vary in their ability to hydrolyse this sugar. The subjects D. A. H. and W. R. G. were apparently still excreting lactose 10 hours after the beginning of the experiment, and when the experiments were repeated almost

11 LACTOSE IN URINE. 351 Sugar after Time of passing Sugar hydrolysis "Lactose" urine (g.) (g.) (g.) W.R.G. 64kg.: _ (took 50 g. lactose) * * *09 0* *15 0* * a.m next day LW.D. 73kg.: (took 50 g. lactose) * a.m next day D.A. H. 65 kg.: ' (took 50 g. lactose) identical results were obtained. The following figures show that in the case of L. B. W. the excretion of lactose is quickly over. Sugar after Time of passing Sugar hydrolysis "Lactose" une (g.) (g.) (g.) L. B. W. 83 kg.: _ (took 50 g. lactose) (took 75 g. lactose)

12 352 L. B. WINTER. Though the taking of 50 g. lactose by the mouth resulted in different amounts of this sugar being excreted by different individuals, all experiments agreed in showing that lactose excretion did not begin until 2 hours after the sugar was taken. Whether after that time lactose was in fact excreted is uncertain, for the quantity was always too small for identification by chemical methods, but the increased reducing power on heating the urine with acid renders it highly probable that traces of unchanged sugar were present. All experiments also showed that the first result of taking lactose is an increased excretion of reducing sugar which is not lactose, since the titration figures were not increased by boiling the urine in the presence of acid. That this sugar is not necessarily galactose is shown by the following experiment. W. R. G. after emptying the bladder received 50 g. lactose at a.m. At 1.30 p.m. 400 c.c. urine were passed containing 0-72 g. sugar (reckoned as glucose, and with the value remaining unaltered after hydrolysis). The sugar was extracted in the usual manner, and 0'33 g. was isolated without difficulty A sample was recrystallized for analysis, and the following figures were obtained. M.P *0 mg. in 201 g. water. c = 09. Initial Final a = [a]54 13 = C 40 04, 40-20; H 6-87, Phenyl-osazone: M.P [a]15 =-81 falling to Individuals vary in their tolerance t.o the galactose half of the molecule. D. A. H. passed urine at and took 50 g. lactose. The next sample of urine (230 c.c.) was passed at 3.0 p.m. and contained 0*41 g. of sugar calculated as glucose; the reducing value of the urine was not raised by heating with acid. The sugar was extracted, but the syrup crystallized only partially, and was drained on a porous tile. The solid was washed with alcohol, dried, and recrystallized from methyl alcohol. 22 mg. of sugar were obtained with the melting point and specific rotatory power of agalactose. M.P mg. in 1-60 g. water. c = Initial Final a = [a] 146 = C 39 77, 39-96; H 6*74, The very small yield of galactose obtained, and the difficulty of separation of this constituent, point to there having been another sugar

13 LACTOSE IN URINE. present, probably glucose; it was not possible however to induce this other sugar to crystallize. Lactose injected into the circulation. Rabbits were used. The animals were placed in a metabolism cage and allowed water and a little cabbage for 24 hours before and during the experiment. The urine was collected during the 12 hours preceding the sugar injection, and the total "sugar" was estimated. Lactose dissolved in 5 c.c. warm saline was then injected into an ear vein, and the sugar excretion during the next 12 hours was determined. The difference gives an approximate figure for the lactose excretion. The variations in different experiments were very wide, particularly when lactating animals were used. In the latter experiments the rabbit was placed with the young in the cage in order not to disturb the normal removal of milk from the breasts; the time selected was 10 days after parturition, when lactation was at its height and the young were just out of the nest. The following are representative experiments. Lactose Weight Injected Excreted Exp. Sex (kg.) (g.) (g.) 1 M F. 3* F. (lactating) ,,,, ,, PI F36 The increased reducing power after heating the urine with acid indicated that in all cases it was the unchanged sugar which was excreted. Separation of the sugar from the urine of the rabbit proved somewhat difficult, and the yield was poor. The following are the details of Exp. 3 in which the lactose was isolated. 130 c.c. urine collected in the 12 hours preceding injection, containing 0 03 g. "sugar." 121 c.c. urine collected after injection of lactose. 5 c.c. were diluted with an equal volume of N HCI; 4 c.c. of the mixture required 19 c.c. of permanganate before and 3-7 c.c. after boiling, giving a "lactose" content of 0*67 g. From 75 c.c. of this urine 70 mg. of sugar were obtained. M.P [a]1461 = + 85 falling to The above experiments show that there is no indication that even the lactating animal is able to use unhydrolysed lactose, but it is clear from the individual variations in either group of experiments that the question of the non-utilization of lactose cannot be settled by relying on urine analyses. All that can be concluded is that when lactose is givein 353

14 354 L. B. WINTER. by the mouth little is excreted, and when injected into the circulation much is passed into the urine, but how much and how little respectively it is impossible to determine. More satisfactory evidence is afforded by experiment based on the following argument. If lactose is utilizable by the tissues there will be a definite threshold for this sugar in the blood; no substance of use to the body would be likely to be allowed to escape through the kidney while it was being transported by the blood to the tissues able to use it. If while lactose is being excreted there is no identifiable amount circulating, then it may be concluded that all of this sugar is promptly excreted as soon as it reaches the blood; if on the other hand there is a threshold for lactose, there will be an appreciable amount present in the blood under these conditions. The results obtained by this line of attack will be given in the second part of this paper, dealing with the examination of the blood sugar during lactation. SUMMARY. 1. During the puerperium women frequently pass urine which contains an abnormally high concentration of reducing substances; lactose is the only sugar which has been separated from such urines. 2. Before parturition it is uncommon for the reducing substances of the urine to be increased; when this occurs the sugar is glucose. 3. Identification of sugar in urine by condensation with phenylhydrazine may be very misleading. 4. The tolerance of normal men for lactose taken by the mouth is considerable. If excretion of lactose takes place after 50 g. doses, the amount is very small and does not occur until 2 hours after taking the sugar. 5. The immediate result of taking lactose by the mouth is an excretion in small amount of either glucose or galactose, or possibly both sugars, depending on the individual. 6. Lactose injected intravenously into rabbits is largely excreted unchanged, irrespective of the sex. This is also true of lactating animals. I am grateful to Dr J. P. Hedley for giving me every facility for obtaining material from his cases, and to the Nursing Staff for their cooperation. My thanks are due to Prof. J. Mellanby, F.R.S., for ensuring that the physiological aspect was not lost sight of during the chemical work. The expenses of this work were in part defrayed by a grant from the Government Grant Committee of the Royal Society.

15 LACTOSE IN URINE. 355 REFERENCES. Folin and Berglund (1922). J. Biol. Chem. 51, 213. Harrison (1930). Chemical Method&. Churchill, London, 94. Hofmeister (1877). Hoppe-Seyl. Z. 1, 101. Lemaire (1895). Hoppe-Seyl. Z. 21, 442. Needham (1924). Biochem. J. 18, 891. Neuberg (1899). Ber. 32, Porcher (1904). C.B. Acad. Sci. Pari8, 138, 833. Rowley (1922). Collected Papers, Mayo Clinic, 14, 459. Svanberg (1930). Hoppe-Seyl. Z. 188, 217. Watkins (1928). J. Biol. Chem. 80, 33. Winter (1925). J. Phy8iol. 80, Proc. p. xxi. Winter (1930). Biochem. J. 24, 851.