John R. Murlin

Hyperglycemia Following the Portal Injection of Insulin.

Conclusion A transient hyperglycemia follows the portal injection of insulin.

A rapid method of preparing the anti-diabetic substance of pancreas

As reported 1 at the last meeting of this branch, heating to 75° and 80° C. for one-half hour does not destroy the anti-diabetic substance extracted in acid aqueous media. The original observation of Murlin and Kramer that a potent extract could be prepared by boiling dogs pancreas in 0.2N HCl was confirmed 2 some time ago. Recently we have compared the yield of rabbit units obtained on the one hand by grinding macerated pancreas in 0.2N HCl in a bacteria grinder for 15 hours and, on the other hand, by heating to 75° C. for one hour or bringing just to boiling temperature. Kimball, Piper and Allen 3 showed also at the last meeting that the active substance is precipitated by complete saturation with sodium chloride, and with either of the alcohols, methyl, propyl, butyl or amyl when added to a solution in 80 per cent. ethyl alcohol. For example, preparation No. 140, heated in 0.2N HCl to 75° C, cooled, strained through cheese cloth, neutralized to PH of 4.1, and filtered, gave in two tests representing 10 gms. pancreas each, a fall of blood-sugar in normal rabbits of 44 and 62 milligrams. The second day the filtrate was precipitated by saturation with sodium chloride, the precipitate taken up in 70 per cent. alcohol, centrifuged to remove insoluble protein and reprecipitated with 5 vols. amyl alcohol. The excess of amyl alcohol was evaporated off before a fan and the residue taken up in sterile water. Equivalent doses representing 10 grams pancreas now gave drops in blood-sugar in rabbits of the same size of 77 and 74 milligrams showing that all the potency had been removed. The filtrate from the NaCl precipitate contains no active substance.

The influence of pancreatic extracts upon the carbohydrate metabolism of depancreatized dogs

The experimental work reported dealt with the effects of extracts prepared in various ways from the pancreas of the dog, pig and ox upon the blood sugar, D:N ratio, respiratory quotient and clinical condition of depancreatized dogs. A dog was given two days to recover from the operation and to become totally diabetic as shown by a D:N ratio of 2.8 or more. The dogs were fed ground beef in increasing amounts after the first day. The extracts were administered by stomach tube, intramuscularly, intravenously, subcutaneously and intraperitoneally. Extract given by stomach tube caused no or very little fall in blood or urinary sugar. Injections of the extract into vein, tissue and peritoneum have been followed by abrupt lowering of the sugar in the blood and urine, the former at times even below normal and the latter has been made to completely disappear even when glucose had been previously administered. In the majority of cases the lowest level of blood sugar was reached in 4 hours from the time of injection whether the extract was given into vein, peritoneum, muscle or under the skin. Since harmful effects were experienced more frequently when the extracts were injected intravenously, intramuscularly and intraperitoneally than when given subcutaneously the last method was used most. The first preparations used in this series of experiments were extracts of freshly removed, macerated dogs pancreas given without further treatment. These proved exceedingly toxic causing extensive sloughing and ulceration of the tissues into which they were injected, and sometimes even caused the death of the dog. These wounds were sterile unless secondarily infected. Simple alcoholic extracts likewise produced toxic effects, particularly when only small volumes of alcohol were used and the trypsin was for this reason not wholly destroyed.

Three months study of the influence of the anti-diabetic substance on a case of severe diabetes

Anti-diabetic substance, prepared at the University of Rochester was given to a case of severe diabetes mellitus (adult) by the duodenal tube, by mouth and by subcutaneous injections. Accurate study was made to determine the potency of the pancreatic extract, the dosage required and the most efficient and practical method of administration. Glycosuria had been present at least for five years and was progressively increasing. Acidosis was constant during the past year. At the time of beginning this study the elimination of urinary sugar was greater than the intake of carbohydrate. The patient was placed in the Rochester General Hospital, July 6, 1922, and studied while on a weighed diet consisting of carbohydrate 29.5 gms., protein 69 gms., fat 107.48 gms., and total calories 1394. During this study the blood sugar averaged from 0.410 to 0.513; the urinary sugar averaged from 32 to 45 grams; and the acetone and diacetic acid was 24. Anti-diabetic extract was given fifteen times through the duodenal tube from July 12, 1922 to July 29, 1922 inclusive. The blood sugar was reduced, reaching its lowest, 0.241 on July 26, 1922. The urinary sugar at this time reached its lowest point, 9.17 gms. Acetone was reduced about one half the former quantity and diacetic acid was absent on fourteen of the eighteen days treatment. The patient was transferred to his home July 31, 1922 and kept at rest and on a diet consisting of carbohydrate 28.52 gms., protein 76.16, fat 127.04, and total caloriles 1,900. No treatment was given from July 29, 1922 to August 13, 1922. During this time the blood sugar rose somewhat but did not return to the level before treatment was started. The urinary sugar averaged from 39 to 52 grams.

The energy metabolism of parturient women

Experiments designed to compare the energy metabolism of mother and child just previous to and immediately following parturition were carried out with the bed calorimeter. Three subjects were secured through the out-patient department of the McLean Lying-in Hospital. They were cared for in the New England Deaconess Hospital near the laboratory and were kept on a carefully regulated diet, which, except for the day of parturition and one or two days thereafter, was essentially the same throughout for each case. Early in the morning before breakfast was taken, the subjects were brought to the laboratory (in an ambulance when necessary) and were placed in the calorimeter for periods of two or three hours during which hourly determinations of the carbon dioxide output, the oxygen absorption, the heat elimination and the heat production were made. The heat production was calculated also by the Zuntz method from the amount of nitrogen in the urine, the carbon in the expired air and the oxygen absorbed. A very satisfactory agreement was found between the two methods.

Depancreatized Dogs Kept Alive Several Months with Insulin Administered by Stomach Tube.

Gaebler and Murlin 1 have found that insulin in ox-blood serum administered to phlorhizinized dogs by stomach tube caused positive, though small, effects on the respiratory metabolism and on the excretion of sugar, indicating combustion of more glucose than was burning just before the insulin was given. Blood serum was chosen because of its anti-tryptic action. 2 The insulin effect was not so great nor so prolonged as had been obtained earlier 3 with a salt precipitate of insulin on depancreatized dogs. Recently a number of dogs totally deprived of the pancreas and fed on meat have been kept alive for periods of six weeks to four months on daily doses of 40 to 100 clinical units of insulin in blood serum administered carefully by stomach tube in such a way as to preclude the possibility of absorption from the mouth. The effect on respiratory metabolism is immediate. Respiratory quotients as high as 1.06 have been obtained and in several instances, as reported with salt precipitates, 3 the capacity to oxidize sugar has persisted for 24 hours and more. Frank, Nothmann and Wagner 4 have observed similarly prolonged effects from the use of their synthetic compound given by mouth. The effect on blood sugar is not so sudden as after subcutaneous or intravenous injection and several times effects on respiratory metabolism were obtained without any measurable effect on blood sugar. Blood serum affords partial protection of insulin against the known destructive action of pepsin in the stomach and from possible destructive action of erepsin in the intestinal mucosa. This is a preliminary report.

The biological value of cereal proteins in human nutrition.

Two different studies, one upon five human subjects, the other upon four, were carried on with a view to discover whether amongst the respective proteins of corn, wheat and oats, as presented to the American public in the form of cereal breakfast foods, any difference in biological value could be detected. In the first study the method of Karl Thomas 1 was followed and the so-called “metabolic nitrogen” determined in control periods of two days each. Alternating with these periods were periods of three days, during each of which the cereals successively supplied nitrogen in amount equal to the total nitrogen excreted in the control periods, or nearly so. The chief objections to the Thomas method were the tendency to diarrhoea during the control periods when the theoretical energy supply was derived solely from cornstarch, heavy cream and sugar, and the difficulty of making such a diet palatable. There was no material difference amongst the three cereals as regards biological value whichever method of calculation was employed. In the second study control periods of three days each alternated with cereal periods of four days each; but instead of a protein-free diet, a milk-cream-fruit diet was used deriving 80 per cent of the nitrogen from milk, 10 per cent from heavy cream and 10 per cent from fruit. The tendency to diarrhoea was negligible and the diet was palatable. In the cereal periods the milk protein was replaced by cereal protein, fruit and cream remaining the same, the total calories being equalized by variations in the amount of sugar and corn starch taken. Wheat products gave slightly better replacement values than oats and corn products.

Significance of change in oxygen absorption after insulin in normal rabbits.

Respiration experiments by the closed circuit method employing an apparatus which gave perfect control checks are reported upon twenty fasting rabbits. Seventeen of these received an injection of insulin subcutaneously which reduced the blood sugar an average of 62 mg. in two hours. One of two basal periods of 45 to 60 minutes each were obtained before giving insulin, and two periods of at least 45 minutes each, in all cases, and four periods in five of the cases, were obtained after insulin. There was no rise but often a fall in respiratory quotient during the first period up to one hour after insulin. In the second period, however, the average respiratory quotient was 0.98 in contrast with the average of 0.74 in the pre-insulin period. In the third period after insulin, the respiratory quotient returns to the normal level and persists also in the fourth period. The oxygen absorption on the average rises the first hour and falls considerably below the pre-insulin level the second hour. The CO2 elimination rises the first hour and the average for all the animals rises still farther the second hour. But the average for the five animals studied longest the CO2 does not rise farteher in the second period, and falls toward but does not quite reach the pre-insulin level in the third and fourth periods. Calculations of the metabolism, by the Zuntz and Schumburg method, of several animals in which the urinary nitrogen was known, shows in the first hour after insulin an average increase amountinig to 16 per cent. In the second period terminating at 1% hours after insulin, there is an ablrupt change in the metabolism from fat to Carbohydrate.

Some favorable effects from the alimentary administration of insulin.

Since July, 1922, when absorption of insulin from the alimentary tract was first observed by us 1 , 2 various attempts have been made to devise a practicable method for oral administration. No difficulty was experienced in demonstrating absorption of simple extracts by depancreatized dogs proving that insulin can survive the destructive action of the stomach. 3 With normal animals, normal human subjects and diabetic patients having an efficient supply of trypsin the insulin was almost always destroyed. 4 , 5 Nevertheless a few instances of obvious absorption were encountered in human diabetics. These were reported in brief at the 11th International Congress 6 of Physiologists at Edinburgh, July, 1923. In this same month attempts were made to delay the destructive action of trypsin by combining with insulin certain weak organic acids and enclosing the combination in enteric coated capsules. Later enteric coated tablets were prepared for us by reputable pharmaceutical houses. Several cases have now been treated for different lengths of time with these tablets. Not all reacted favorably. Two of those giving the best reactions are shown in the table together with several additional tests by duodenal administration not previously reported. In all of the four cases reported the patients were on constant diets. The results show : (1) that insulin contained in 0.1 or 0.2 per cent HCl is absorbed from the human intestine; (2) that addition of alcohol to 5 to 8 per cent increases the absorption somewhat; (3) that alcohol alone, however, produces a drop in blood sugar but only a slight effect on the 24 hour elimination of sugar by the kidney; (4) that insulin combined with a weak organic acid or acid salt, the whole contained in an enteric coating which survives the stomach for at least three hours, can be absorbed in sufficient amount to lower blood sugar and urine sugar very materially.

Further observations on the chemical and physical properties of insulin

Probably all active workers with insulin have observed considerable variation in the potency of the final product by whatever method obtained. Experience in this laboratory had led us to reinvestigate especially the influence of the exact H ion concentration upon potency at the several stages of the aqueous method. 1 It has been found that after thorough extraction in N/5 HCl neutralizing with N/1 NaOH to PH of 5.0 to 5.7 gives the largest yield of potency in the first filtrate. The reaction is controlled both titrometrically to phenolphthalein and electrometrically (error ±0.2PH). Stopping at a PH of 4.0 to 4.4 gives more rapid filtration but not so much available potency at once. However if the reaction after filtration is at once readjusted to 5.3 or thereabouts the potency is rendered available or, if it be allowed to stand for 24 hours at PH 4.0 to 4.4 the potency increases. In fact the filtrate may be kept at room temperature until there is an abundant growth of bacteria and yeasts without destroying the potency. In the final concentration it has been found that potency is best preserved in a fairly acid medium .06 to .08 N HCl; though a reaction as high as PH 5.7 preserves for several weeks if an antiseptic like tricresol is added at once. Pasteurization temperature maintained for half an hour does not destroy the potency. In fact the concentrated extract purified of proteins can be heated to 80° for half an hour, the exact effect depending upon the reaction. At a PH of 6.2 to 7.0 or higher heating to this point seems regularly to increase the potency on rabbits. Heating at a PH of 4.4 to 5.7 the potency is usually diminished or carried down with the coagulum if proteins are present.