ON THE MODE OF PRODUCTION OF LACTOSE IN THE MAMMARY GLAND. BY D. NOEL PATON AND E. P. CATHCART. (From the Physiologicat Department of the University of Glasgow.) THE mode of production of the disaccharid lactose in the mammary gland is a problem which has not yet been solved. As is well known, upon hydrolysis lactose splits into a molecule of dextrose and into a molecule of galactose, a sugar whicb is not found free in the body. The idea that the galactose is derived from the galactosides of vegetables,hardly requires consideration since lactose is the sugar of the milk of carnivores as well as of herbivores. The view of Paul Bertl that a glycogen-like precursor of lactose exists in the mammary gland is without foundation, and Thierfelder's theory that the gland contains an enzyme which develops lactose from some precursor also rests upon no substantial basis. P. Mayer2 failed to find in the decomposition products of the proteins of the gland any substance from which galactose might be formed. Baschs was unable to demonstrate the presence of an enzyme capable of producing a synthesis of glucose and galactose. Cremer' advanced the theory that the gland possesses the power of changing glucose into galactose for the formation of lactose, a view as to the mode of action of the gland which has been supported by Porcher5. The gland must either form the lactose independently of the store of carbohydrates in the body, or it must utilise the preformed carbohydrates through the glucose of the blood. It mav do so by forming 1 Gaz. Mdd. de Paris, 1879, No. 12, discussed by Bas oh, Ergeb. d. physiol. Bioch. Ii. 2 Jhrg. p. 375. 1903. 2 Deutsche med. Wochensch. p. 6. 1899. 3 Ergeb. d. physiol. Bioch. ii. p. 375. 1903. 4 Ztsch. f. Biol. xxxi. p. 183. 1895. 5 Arch. Internat. de Physiol. viii. p. 356. 1909.
180 D. N. PATON AND E. P. CATHIART. the lactose entirely from this source or by using the glucose to synthesise with the galactose produced as a specific product of the gland. Attempts to solve the problem have been made by removing the mammary gland from pregnant animals -in order to find if after parturition dextrose appears in the urine, a phenomenon which might be taken to mean that the glucose formed in the liver or elsewhere in the body is used by the mammary gland in the formation of lactose. But the results recorded have been most contradictory and no definite conclusion can be drawn. Thus Paul Bert, from his experiment of removing the mammary gland in two pregnant goats and getting a reducing substance in the urine after parturition, concluded that the dextrose of the blood is used by the gland in the formation of lactose. Moore and Parker2 repeated these experiments with negative results. Porcher8 on the other hand found a large amount of glucose in the urine of goats without the mammary gland and concluded that the animal changes the glucose of the blood into lactose. More recently Marshall and-!-kirkness' repeated the experiment on guinea-pigs with negative results. This method does not appear to us to promise results of value, since it seems probable that the increased sugar production in the body for the preparation of lactose in the gland may be determined by the growth of the gland itself, possibly through an internal secretion, and that it may not make itself manifest in the absence of the gland. It seemed probable that light might be thrown on the question by clearing out to a greater or less extent the available supply of glucose by the administration of phloridzin. The paper of Fichera6 which attempts to prove a glycogenetic action of phloridzin upon the tissues appears to be so inconclusive that it need not be considered in face of the definite evidence that phloridzin does cause the elimination of sugar. If the glucose of the body is the source of the glucose used by the gland for the formation of lactose, this procedure should either markedly decrease the formation of lactose, or it might possibly cause the appearance of galactose in the milk. The further question of whether phloridzin causes the appearance of lactose in the urine of animals during milk secretion or whether it may manifest an action on the mammary gland similar to that exercised on the kidneys seemed to us 1 C. R. de Z'Acad. d. Scien. xcviii. p. 775. 1884. 2 Amer. Journ. of Physiol. iv. p. 239. 1900. 3 C. R. de V'14cad. d. Scien. cxxxvm. p. 833. 1904 4 Biochem. Journ. ii. p. 1. 1906. 5 Ziegler's Beitr. xxxvi. p. 292. 1904.
LACTOSE AND MAMMA RY GLAND. worthy of observation. Since our experiments were completed we find that the same method has been employed by others. Cornevinl injected 10 and next-day 20 grs. of phloridzin in a Normandy cow and records a great increase in amount of sugar per litre of the milk. Pappenheim2 records in the goat a marked fall in the output of milk and of lactose under the administration of phloridzin and states that the sugar of the milk gave no fermentation and that it is therefore lactose alone and further that in the urine glucose alone appeared. The possible appearance of galactose does not seem to have been considered. Cremer3 records a fall in the milk secretion and a fall in the total sugar and he concludes that phloridzin has no special action on the cells of the mammary gland and that its action on the kidneys is without analogue. Porcher4 gives experiments on a cow in which the administration of 20 gm. of phloridzin did not materially modify the output of sugar. In his most recent paper5 Porcher reviews his previous work. He concludes that Cornevin is wrong as to the marked increase in the milk sugar per litre following the administration of phloridzin, and points out that there may be a notable diminution in the amount per diem which he ascribes to the decreased milk production. This he believes is due to the diuresis, but in our experiments diuresis was not present. He further concludes that phloridzin simply causes a glycosuria without lactosuria in milking animals. Our experiments were carried out upon a goat in milk, weighing about 30 kilos.- Methods. The goat was kept in a stall specially constructed to allow of the collection of urine. It was milked twice daily at 9 a.m. and at 5 p.m. The chemical methods which we employed were: total nitrogen by Kjeldahl's method; ash by the ordinary ignition method; fat by the Leffmann-Beam method with special small tubes; and lactose by the method described by Shimidzu, in which the protein is removed by the Ritthausen-Munk method and the sugar estimated by the Pavy-Kumagawa-Suto method. This method we found to yield very good results. It is not very troublesome to carry out. The analyses were done in duplicate. Results. In order that the withdrawal of glucose might be made to manifest its influence the supply of carbohydrates in the food was cut down to a low level. This was only possible by limiting the supply of 1 C. B. de l'acad. d. Scien. cxvi. p. 263. 1893. 2 Arch. f. Verdauungs Krankht. in. p. 421. 1898. 3 Ztsch. f. Biol. xxxvii. p. 59. 1899. 4 C. R. Acad. Sc. cxxxiii. p. 1457. 1904. 5 Arch. Internat. de Physiol. viia. p. 356. 1909. 181
182 D. N. PA TON AND E. P. CATHCART. food, and at once the difficulty presented itself that reducing the diet reduces the milk production and the output of lactose-in the same proportion. Hence we have not so far succeeded in completely depleting the supply of glucose available to the mammary gland. All we have been able to do is to reduce it. The results have been quite distinct. They are shown in the following records of experiments. Exp. I. June 1909. The goat was fed upon a low diet of newly cut grass, about 350 gms., and the amount of phloridzin given, 1S5 gms., was small. Milk TABLE I. Urine Amun Day of Amount Lactose Amount colced collected Sugar as 0/0 Exp. in c.c. amount in gms. in c.c. dextrose present 1 1110 31x18 - - 2 540 15.58* 600 5'76 (total) 3 420 11.59* 550 1P76,, 4 670 21-58 200 None 5 890 29-96 - * 1-5 gis. phloridzin given at beginning of these days. The milk is the milk of the preceding evening's milking and the milking of the morning of the date given. The urine is the urine of the 24 hours to 10 a.m. on the date given. Exp. IL October. The goat had for first three days a full diet of oats 750 gms., hay 300 gms., turnips 1600 gms. For the following six days oats 375 gms., hay 300 gms., turnips 800 gms., and for the last two days the full diet as given above. It was calculated that the second diet yielded 347 gms. of available carbohydrate and fibre. Three grams of phloridzin were injected on the sixth and seventh days. Before this the urine when tested for sugar gave negative results. Milk TABLE II. Urine Lactose, Sugar as dextrose Day Amt. in c.c. amt. in gms. Nature Amount of Exp. per diem per diem of diet collected Per cent Total 1 362 15-93 Full -- - 2 365 14-20,, - 3 355 13-95,, - 4 230 9 04 Low 5 238 9-52,, _ 6 238* 9-26,, 150 1-12 1-68 7 218* 8'26,, 400 5.55 22-20 8 170 6-49,, some loss 7-14 - 9 212 8-71,, 160 4-54 7-26 10 247 10-45 Full - trace only 11 365 13-03,, - - - * Three gms. phloridzin given at beginning of these days.
LACTOSE AND MAMMARY GLAND. 183 In spite of the small dose of phloridzin the effect is quite marked, a fall in the amount of milk and in the output of lactose taking place. On the first phloridzin day the urine yielded almost entirely lactosazone with phenylhydrazin, on the next day mostly glucosazone with a small amount of lactosazone. On the third day there was fermentation but no glucosazone crystals were found. The experiment again shows a marked fall in the milk secretion and in the lactose formation. On the first day of phloridzin the urine gave lactosazone and somne glucosazone crystals. On the second day the glucosazone crystals predominated and on the day after the second dose of phloridzin, when the amount of sugar reached 7K14 p.c., the presence of lactosazone crystals was very doubtful. Exp. III. The goat's food consisted of a full diet for two days consisting of oats 745 gms., hay 300 gms., turnips 800 gms., followed for eight days by a low diet of oats 300 gms., bran 50 gms., hay 250 gms., turnips 800 gms. In this there were about 313 gmis. available carbohydrate and fibre. On the third and fourth days of this diet, four gms. of phloridzin were injected. On the last day of the experiment the full diet was again given. Milk per diem TABLE III. Urine Amt. Sugar as dextrose Nitrogen Day Amount Lactose Fat Ash Nature collected It of Exp. in cc. in gms. in gms. in gms. of food in c.c. Percent. Total Per cent. Total 8.11.10 230 7-13 2-3 1P91 Full 9 252 9 37 4-15 2-14,, - - - 10 324 12-7 3-24 2-72 Low - - - 11. 256 10-14 2-56 2-52 12 251 10 11 4-01 2-16,, - - 13* 205 8-12 0-615 1P76,, Urine lost - 14* 160 6A40 3-36 1-55,, 275 5-71 15-7 1P82 5 005 15 151 5-81 6-5 1-16,, 320 6-88 21-4 2-15 6-88 16 196 7-64 6-8 1-78,, 460 2-10 9-66 1P48 6170 17 230 8,85 4-6 1P63,, 400 - - 1P078 4 31 18 230 9-20 5-3 2-09 FuH 260 1-288 3*34 19 - - - 350-1-806 6-31 * At the beginning of each of these days four gms. phloridzin were injected. In this experiment the fat and ash of the milk were determined by Dr Findlay and the nitrogen was also determined. While the ash and total nitrogen varied directly with the amount of milk and lactose, the fat showed a very marked rise upon the day when the lactose reached
184 D. N. PATON AND E. P. CATHCART. its lowest level and upon the succeeding day when cretion of sugar occurred in the urine (Chart I). the largest ex- LArox &;,. -. e;1e. - ++4@ F44...... e. WA O.r. - -.....4 t I 10 WI.t is Da.'fs. Chart I. Examination of the urine showed, upon the second day of phloridzin, glucosazone and lactosazone crystals, upon the next day a smaller proportion of lactosazone and upon the next again no recognisable lactosazone crystals. After fermentation the percentage of sugar (lactose) left expressed as dextrose was 14. 11. 10 0-308. 15 0-293. 16 0.180. That this was lactose is indicated by increase in the reducing power after hydrolysing to about double the previous figure: 14.11.10 15 16 Direct estimation after ferment -308 *293 *180 The endpoints of the reactions were indistinct. Estimation after further hydrolysis.77-526 *356
LACTOSE AND MAMMARY GLAND. 185 After Exp. III. the goat was milked only once daily at 12 o'clock, and was kept upon full diet from Nov. 19th to Dec. 5th. The surprising result obtained with the fats in Exp. III. induced us to do another experiment. In kind the results obtained resemble those of the previous experiments, but in extent they are much more marked. The amount of Exp. IV. On the 6th and 7th, four gms. of phloridzin were given and on the 7th a little bleeding was observed through the needle of the syringe. On the 5th the goat was put upon a diet of bran, hay and turnips. On the 10th, 300 gms. of oats were added, but these were not given after the 13th. The secretion of urine stopped on the 13th and cabbage leaves were added to the diet. Date 6.12.10 7 8 9 10 11 12 13 14 15 16 171 181 191 20 21 22 23 24 25 6.1.11 9 11 12 13 15 18 Amount in ccm. 200* 270* 135 180 85 65 190 250 285 330 300 Not measured,, 350 325 310 350 375 215 160 150 Milk per diem A -% Lactose in grm. 7-80 11'07 5*26 7'02 3-06 2-14 5-70 9.00 TABLE IV. Fat in grm. 2-2 3-2 9.45 3-42 3.74 3*90 8-36 8'50 6-00 4-90 7.44 8,40 7-50 10-32 Urine per diem Sugar as Amount dextrose Nitrogen in ccm. in grm. in grm. 260 9-60 2-73 190 7'29 1-66 435 78-30 9 75 250 28-50 3'60 165 18-80 4'55 85 7'00 1-83 0 0 0 175 11-09 3 07 830 14-00 5'88 640 8-20 - Notfullv - collected,, 200 180 49 0 93 5'88 7,88 Osazone crystals Gilucose Lactose + - 0 0 160 156-3-60 60 0 + -_- - 0 + + + - - 0 + 1 Goat was allowed out of stall for several hours each day.
186 D. N. PA TON AND E. P. CATHCART. sugar in the urine rose to no less than 78 gms. on the 9th, i.e. in the urine passed on the 8th, while the nitrogen rose from 2-5 gms. to 10 gms. But the most interesting point was that the goat continued to excrete dextrose till -Jan. 9th, i.e. for more than a month. After this glucose was absent from the urine although traces of lactose were present till the 14th. We are not aware of any other instance of the persistence of a glycosuria of over a month's duration following the administration of a dose of phloridzin.. It is to be regretted that no examination of the amount of glucose in the blood was made. FZ 11.;_i = 4I.d'. /0., 4, S.,., &. Chart II. The urine gave the reaction of phloridzin with perchloride of iron till the 11th and on the 12th and 13th a trace of proteins coagulated by heat was present. The milk fell from 200 cm. to 65 cm. The lactose fell from 8 gm. to 2-3 gms. a fall out of proportion to the fall in the amount of milk, while the fat showed an initial rise to over 9 gms. on the 8th, and a later and more sustained rise to 8 gms. on the 12th and 13th (Chart II). The fat was again determined on the 16th, 19th and 21st, 22nd and 23rd, and on Jan. 6th and 12th. The lowest figure of the series was
LACTOSE AND MAMMARY GLAND. 4-9 on Dec. 19. With the disappearance of glucose from the urine after Jan. 9th the figure fell to 3-6. The enormous excretion of sugar in the urine on the 9th followed by the very marked fall in the sugar of the milk on the 10th and 11th, led uis to consider whether possibly in this case a failure to completely synthesise the lactose of milk had occurred. Unfortunately only 200 cm. of the diluted protein free milk remained, equivalent to 10 c.c. of milk. This had been evaporated and preserved, and it was re-dissolved in 100 c.c. of water and a direct determination of the lactose made without hydrolysis. The result should, of course, correspond with the calculated results previously arrived at after hydrolysis, if lactose is the only sugar present. The following results were obtained: After hydro- Amount required Before hydro. Iysis c.c. of if all lactose and lysis c.c. of Date milk sol. used not hydrolysed milk sol used 7.12.10 10-6 20-1 19-9 8 11-0 20-9 22-2 9 11-1 21-1 22-0 10 12-0 22-8 23-8 11 1381 24-9 21-8 12 14-7 - The fact that whereas on the 7th, 8th, 9th and 10th the observed reduction of the non-hydrolysed sugar corresponds with or is rather lower than that of the calculated figure for lactose, on the 11th it is actually 3 c.c. greater suggests the possibility of the presence of a mono-saccharid along with the lactose. The amount of fluid at our disposal was too small to enable us to further investigate this point. CONCLUSIONS. These experiments show that, while under phloridzin there was no marked increase in the amount of urine secreted, the amount of milk fell Low diet Phloridzin Exp. 1I 235 to 170 Exp. III 256 to 151 Exp. IV. 200 to 65 The lactose of the milk fell Low diet After phloridzin Fall Exp. II 9-25 6-49 2-76 Exp. III 10-14 5-81 4-33 Exp. IV 9 4 2-14 7-3 187
188 D. N. PATON AND E. P. CATHCART. The lowest output being reached either on the day that the largest output of sugar occurred in the urine or just after this. These experiments -indicate that the glucose of the blood is used by the mammary gland in the formation of lactose. They leave unsolved, however, the question of the source of the galactose. The fact that, despite the decreased formation of lactose, sugar appears in the urine seems to us to hint that there is a limitation in the supply of a precursor common to both its constituents, and tq suggest that the glucose of the blood is probably the forerunner of the galactose as well as of the dextrose of milk sugar. The only hint of a possible independent source of galactose is to be found in the result of the sugar determination in the milk of the 11th in the last experiment. This point we intend to investigate further next summer. The conclusion that the blood glucose is the source of the lactose is supported by the observations of Porcher that before delivery dextrose may appear in the urine in the human subject-but that after delivery when sugar appears it is lactose. Our observations also indicate that phloridzin increases the permeability of the kidney for lactose as well as for glucose. 'The increase in the fat of the milk indicated by the last two experiments is of very special interest. Rosenfeld2 showed that in fasting animals, the administration of phloridzin causes a great increase of fat in the blood and in the liver. Is this another indication that for the utilisation of fats the presence of carbohydrates is necessary, and is this increase in the fat of the milk an expression of a non-metabolism of fat in the mammary gland the result of the withdrawal of sugar? (The expenses of this investigation were defrayed from a grant from the Carnegie Trust.) 1 Just before sending off this paper we have found the record of a single experiment on the influence of phloridzin on the fat of the milk of the goat by Graham Lusk (Ztsch. f. Biol. xl. p. 42. 1901). He finds a marked increase in the percentage amount of fat but a decrease in the total amount. 2 Ergeb. d. phys. Biock. ii. p. 70. 1903.