PLASMA AND URINARY AMINO-ACIDS IN DIABETES

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1 PLASMA AND URINARY AMINOACIDS IN DIABETES By C. H. GRAY and ELIZABETH K. B. ILLING From the Department of Chemical Pathology, King's College Hospital, Denmark Hill, London, S.E. 5 (Received 22 June 1951) SUMMARY The plasma concentration of \g=a\aminon was determined in fiftythree diabetic patients. These diabetic patients included cases with and without ketosis, cases before and after insulin treatment, and cases whose diabetes started before and after the age of 40 years. None of the patients with ketosis was in coma or precoma. No significant difference from the normal plasma \g=a\aminon concentration was observed in any of these six groups. Qualitative chromatographic analysis and quantitative determinations of the aminoacid excretion of thirtythree and fourteen diabetic patients respectively, showed a significantly increased excretion of amino N during diabetic ketosis. This increase may be associated with an excess production of free aminoacids due to an increased secretion of adrenocorticotrophic hormone during diabetic ketosis. The adrenocorticotrophic and growth hormones of the anterior pituitary influence the blood aminoacids of hypophysectomized and normal animals and produce a diabetic state in the experimental animal and in the human subject. A study of the aminoacid concentration in blood and urine in the various clinical types and stages of diabetes might be expected to throw some fight on the nature of the endocrine abnormality in this condition. Previous reports concerning the aminoacid concentration in the blood of diabetics are conflicting. Von Falkenhausen [1924], Greene, Sandiford & Ross [1924], Witts [1929] and Schmidt [1929], all of whom used Folin's [1922] colorimetrie method of determination, reported values within normal limits. Wolpe [1924], using the same method, reported a concentration above normal ; three of his six cases were in coma or acidosis. Desqueyroux [1923], using the Sorenson [1907] titrimetric method, found considerably raised concentrations in two of six cases. Feinblatt & Shapiro [1924] observed a high concentration in only one out of twenty patients; they used the Folin method. Luetscher [1942], using the specific and accurate ninhydrin method of Van Slyke, Dillon, MacFadyen & Hamilton [1941], observed high concentrations of plasma aminoacid in all of twelve severely ketosed untreated diabetic patients. Normal concentrations were observed after treatment with insulin. Medrano [1944], with Folin's method, found a normal concentration in the blood of diabetic patients without complications but a raised concentration in diabetics with acidosis. Galambos & Tausz [1913] found an increased urinary excretion of aminoacids in severely diabetic patients. Luetscher [1942] also observed an increased excretion of aminoacids returning to normal with treatment in his twelve diabetic patients with ketosis. Hall [1948] reported a marked aminoaciduria in twenty of eighty diabetic patients, whose urines were examined by paper partition chromatography. It appeared of interest to reinvestigate the concentration of aminoacids in the blood of diabetic patients, to attempt to confirm Hall's findings and to extend them to determine whether the aminoaciduria was related to the type or stage of diabetes.

2 AMINOACIDS IN DIABETES 45 METHODS Most of the fiftythree diabetic patients studied were ambulatory outpatients attending the Diabetic Clinic at King's College Hospital, although a few had been admitted to the ward for stabilization. Plasma was separated from blood samples collected into oxalate by vein puncture from the antecubital vein between 10.0 a.m. and p.m. (at least 2 hr. after breakfast). Specimens showing haemolysis obvious to the naked eye were discarded. Plasma was stored at 0 as such or after precipitation with picric acid ; under these conditions the plasma amino is unchanged for 14 days [Hamilton & Van Slyke, 1943]. Urine specimens were preserved at 0 under toluene or with thymol and were collected under the supervision of a competent sister and can be relied upon as being accurate 24 hr. collections. Plasma and urine aminonitrogen were determined by the method of Hamilton & Van Slyke [1943]. Blood urea and apparent blood sugar were determined by the methods of Barrett [1935] and Folin & Wu [1920] respectively. Chromatographie analysis of urinary aminoacids. The 24 hr. urine collections were diluted with water to 3000 ml. to facilitate a rough quantitative comparison of aminoacid excretion. The varying and very large quantities of glucose in diabetic urine invalidated the customary procedure of dilution to a given specific gravity. 010 ml. of diluted urine was used on onedimension strips with phenol as solvent in the presence of ammonia [Dent, 1948]. After about 20 hr., giving a run of about 30 cm., the strip was dried overnight at room temperature and sprayed with 01% ninhydrin in Mbutanol. The urines were not desalted before chromatography. Five rather large spots were usually distinguishable corresponding to (a) aspartic and glutamic acids, (6) serine, glycine and taurine, (c) alanine and glutamine, (d) valine and lysine, and (e) leucine. The intensities of the spots were roughly assessed by the method of Dent & Schilling [1949] by which the spots are compared with a colour chart given by the ninhydrin reaction on filterpaper of ten different amounts of aminoacids. The intensities were then numbered 13, 47 or 810, according to whether the spots were faint, moderately strong or strong. RESULTS Table 1 presents the aminoacid concentrations found in the plasma of the fiftythree diabetic patients, together with the corresponding values for normal persons, which agree well with those of Woodruff & Mann [1945]. There was no significant difference between the results with diabetic patients and those from normals, nor between those from diabetic patients with or without ketosis or from insulintreated diabetics and from diabetics not treated with insulin. The results from patients whose diabetes began below the age of 40 did not differ from those of patients whose diabetes began after this age. The range for the diabetic patients is somewhat greater than the normal range, three patients showing values below 337 and twelve above 497 mg./100 ml. plasma, the upper limit of normal. There was no obviously different incidence of patients with ketosis, insulintreated patients, or patients with diabetes developing late in life in these fifteen patients, as compared with the remainder.

3 46 C. H. GRAY AND ELIZABETH. B. ILLING Table 1. No. of Group observations Diabetic 53 Normal 5 Normal [Woodruff & Mann, ] Diabetic with ketosis 21 Diabetic without ketosis 32 Untreated diabetic 21 Treated diabetic 32 Diabetic under 40 yr. at onset 29 Diabetic over 40 yr. at onset 23 Plasma aminonitrogen in diabetic patients mg. amino /100 ml. plasma Mean + s.k. 448 ± ± ± ± Comparison of groups Diabetic/normal 066 Diabetic with ketosis/diabetic without ketosis 028 Untreated diabetic/treated diabetic 056 Diabetic under 40 yr. at onset/diabetic over 40 yr. at onset 090 Table 2. Group Diabetic with ketosis Diabetic without ketosis Diabetic with ketosis Diabetic without ketosis Fisher's t Range Probability that difference is due to chance ^03 Correlation of plasma xamino concentration with concentration of (a) blood urea and (b) apparent blood sugar Mean plasma Mean blood amino concentra No. of tion tration N concen cases (mg./100ml.) Range (mg./100ml.) Range (a) Blood urea concentration (6) Apparent blood sugar concentration Correla tion coeffi cient > >01 Table 2 and Figs. 1 and 2 show that there was no correlation between the plasma aminonitrogen and the blood urea or the apparent blood sugar. Fig. 3 shows the results of qualitative Chromatographie analysis of thirtythree 24 hr. urine specimens from twentysix diabetic patients as well as of urine from six normal persons. Rough quantitative assessment of the intensities of the spots obtained showed that in ten of the samples there was a gross increase in the amount of aminoacids present probably due to an increase in all five groups of aminoacids investi gated rather than in only one or some of the groups. Of the ten samples showing the gross aminoaciduria, seven contained acetoacetic acid. Since only twelve of the samples as a whole contained acetoacetic acid this represents 63 % of the urines from patients with ketosis, whereas only 14 % of the urines from patients without ketosis showed obvious excessive aminoaciduria. Six patients were studied, both with ketosis and later when the ketosis had disappeared; two of these showed no excess aminoaciduria either before or after treatment of the ketosis. The aminoaciduria

4 AMINOACIDS IN DIABETES 47 present in the other four patients disappeared with treatment in three but persisted despite disappearance of ketosis in the fourth patient. Table 3 presents the results of quantitative determination of the aminoacid excretion in fourteen diabetic patients together with the corresponding values for = J L XX XX > Plasmaotamino N concentration (mg./l00 ml.) Plasma amino concentration (mg./100 ml ) Fig. 1. Fig. 2. Fig. 1. Relationship between blood urea concentration and plasma amino concentration. > diabetics with ketosis ;, diabetics without ketosis. Fig. 2. Relationship between apparent blood sugar concentration and plasma amino concentration, diabetics with ketosis ;, diabetics without ketosis. Table 3. Group Diabetic Normal Normal [Thompson & Abdulnabi, 1950] Diabetic without ketosis Diabetic with ketosis Diabetic under 40 yr. at onset Diabetic over 40 yr. at onset Urinary aminonitrogen in diabetic patients No. of observations mg. urinary amino N/24 hr. Mean + s.e. 2010± Comparison of groups Diabetic/normal 25 Diabetic with ketosis/diabetic without ketosis 26 Diabetic with ketosis/normal 29 Diabetic without ketosis/normal 09 Diabetic under 40 yr. at onset/diabetic over 40 yr. at onset 16 Fisher's t Range Probability that difference is due to chance

5 48 C. H. GRAY AND ELIZABETH. B, ILLING a small series of normal persons. These latter are in agreement with those of Thompson & Abdulnabi [1950]. The urinary excretion of amino acids per 24 hr. is significantly greater in diabetic patients than in the normal subject. Eight of the diabetic patients had no ketosis and the urinary amino acid excretion in those cases was not significantly different from that of the normal; that of the remaining six patients with ketosis was greatly increased. Fig. 3. Paper partition Chromatographie analysis of aminoacid in normal and diabetic urines., aspartic and glutamic acids; \J ], serine, glycine and taurine; fj, alanine and glutamine; g, valine and lysine; ^, leucine. Ordinate = intensity of spots in arbitrary units, a = before treatment, 6 = after 1 week of insulin treatment. DISCUSSION It is clear that the aminoaciduria present in some cases of diabetes appears to be unrelated to the type or stage of diabetes but to accompany ketosis. Low plasma concentrations of aminoacids were not observed, so that these results cannot be explained on the basis of a lowered renal threshold. However, none of the patients in the present series showed an increase in the aminoacid content of the blood,

6 AMINOACIDS IN DIABETES 49 although Luetscher [1942] found high plasma levels in all of twelve patients with severe ketosis. Since Luetscher also used the specific ninhydrin method, the dis crepancy between his and our results is probably because the ketosis was more severe in his patients. The increased excretion of aminoacids must be due either to increased production or to decreased utilization. There is no evidence of this latter possibility, for in Luetscher's patients the urea excretion was not low, nor was the high concentration of aminoacids in the plasma related to renal insufficiency. Ketosis thus seems to be associated with the production of free aminoacids in excess. These are usually excreted by the kidney, but if the ketosis is so severe that the kidney is unable to excrete them sufficiently rapidly, the concentration in the blood increases. The blood aminoacids of hypophysectomized animals are increased and decreased by the adrenocorticotrophic and growth hormones respectively of the anterior pituitary. The quantity and concentration of aminoacids in body fluids must depend not only upon the total intake and urinary output but also upon the reversible equilibrium with body.protein, presumably under the action of growth hormone, as well as upon the utilization process apparently mediated through adrenocorticotrophic hormone. In fasting diabetic patients, therefore, the increased concentration of aminoacids in the body fluids could be due either to a diminished activity of growth hormone or to an overaction of the adrenocorticotrophic hormone. Since ketosis is accompanied by increased excretion of adrenocortical hormones [McArthur, Sprague & Mason, 1950], it is possible that the increased production of aminoacids observed in patients with ketosis may be related to excess adrenocorticotrophic activity of the anterior pituitary. We wish to express our thanks to Mr H. E. R. Millar for collecting the blood samples and to Messrs Boots for a personal grant to E.K.B.I. REFERENCES Barrett, J. F. [1935]. Biochem. J. 29, Dent, C. E. [1948]. Biochem. J. 43, 169. Dent, C. E. & Schilling, J. A. [1949]. Biochem. J. 44, 318. Desqueyroux, J. [1923]. Ann. Med. 13, 20. Falkenhausen, M. F. von [1924]. Arch. exp. Path. Pharmák. 103, 322. Feinblatt, H. M. & Shapiro, I. [1924]. Arch. Intern. Med. 34, 690. Folin, O. [1922]. J. Biol. Chem. 51, 377. Folin, O. & Wu, H. [1920]. J. Biol. Chem. 41, 367. Galambos, A. & Tausz, B. [1913]. Z. klin. Med. 77, 14. Greene, C. H., Sandiford, K. & Ross, H. [1924]. J. Biol. Chem. 58, 845. Hall, D. A. [1948]. Biochem. J. 43, lviii. Hamilton, P. B. & Van Slyke, D. D. [1943]. J. Biol. Chem. 150, 231. Luetscher, J. A. [1942]. J. Clin. Invest. 21, 275. McArthur, J. W., Sprague, R. G. & Mason, H. L. [1950]. J. Clin. Endocrinol. 10, 307. Medrano, E. [1944]. Medicinia, 24, 162. Schmidt, E. G. [1929]. Arch. Intern. Med. 44, 351. Sorenson, S. P. L. [1907]. C.R. Lab. Carlsberg, 7, 1. Thompson, R. C. & Abdulnabi, M. [1950]. J. Biol. Chem. 185, 625. Van Slyke, D. D., Dillon, R. T., MacFadyen, D. A. & Hamilton, P. [1941]. J. Biol. Chem. 141, 627. Witts, L. [1929]. Quart. J. Med. 22, 477. Wolpe, G. [1924]. Munch, med. Wschr. 71, 363. Woodruff, C. W. & Mann, E. B. [1945]. J. Biol. Chem. 157, 93.