Knowledge of the subjects' previous dietary history, daily activity, and

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1 NORMAL VARIATIONS IN TOTAL KETONE BODIES IN SERUM AND URINE OF HEALTHY YOUNG MEN. By R. E. JOHNSON, F. SARGENT, II and R. PASSMORE. From the Departments of Physiology, University of Illinois, Urbana, U.S.A. and Edinburgh University. (Received for publication 22nd March 1958) To establish normal values and ranges for studies of ketosis, total ketone bodies have been estimated in the urine and serum of 208 healthy young men, eating non-ketogenic adequate diets and engaged in moderate daily physical activity. Under standardized post-absorptive conditions timed specimens of urine were collected, and venous blood was drawn. The urinary excretion rate of ketone bodies differed significantly in summer and winter. In hot weather the mean rate was 0 9,c-mole/min.; in cold weather it was 2-8 ju-mole/min.; in cool weather it was 1X7,c-mole/min. For serum, regardless of season, the mean was 0 7 m-mole/l. If two standard deviations be taken as the normal range, it is suggested that for most conditions the upper limit of normal for serum be considered as 1-4 m-mole/l. and for urine 5 u-mole/min. AN interest in ketosis which may arise in healthy men from nutritional causes, especially starvation, and after prolonged exercise (the Courtice-Douglas effect), led us to inquire: "What are the normal values for total ketone bodies in blood and urine? " A systematic study of changes in ketone metabolism in man demands knowledge of the normal, yet present information is scanty and unsatisfactory. For instance, the Handbook of Biological Data [Albritton, 1953] has no separate lists for the individual ketone bodies in urine, and only gives the total excretion in daily specimens collected at random without regard to previous dietary history or activity of the subjects. Furthermore, analytical methods have been unsatisfactory and tedious. For betahydroxybutyric acid, which may account for half or more of the total ketone bodies, no qualitative test exists, and hitherto many quantitative methods have not given true values for it. During metabolic studies with 208 subjects, we have been able to collect specimens of blood and urine under standardized post-absorptive conditions and to analyze these with satisfactory quantitative and qualitative methods. Knowledge of the subjects' previous dietary history, daily activity, and environment was adequate. Hence, we are able to assert that for healthy young men we know the normal range of concentration of total ketone bodies in serum; the normal rate of excretion of these substances in urine under post-absorptive conditions; and the relation between quantitative and qualitative examination of urine collected under these conditions. Qualitative results have been reported by Sargent and Consolazio [1950] which support VOL. xliii, No

2 340 Johnson, Sargent and Passmore the view that exposure to cold increases the rate of excretion of acetone plus acetoacetic acid. We have confirmed this observation quantitatively for total ketone bodies. METHODS Subjects.-Three studies have been conducted: summer, 1955 (Indiana); winter, 1954 (Wisconsin); and winter, 1957 (Edinburgh). In each of the first two, ninetynine healthy young airmen were the subjects, and in the last, ten healthy young medical students. Activity and Environment.-Daily work was moderate, consisting of walking outdoors to and from classes, other duties and meals. At most other times the subjects were living a sedentary life and were not much exposed to the weather, even though the outdoor temperatures were rather severe. TABLE I.-SUBJECTS AND REGIMENS Duration of Total Percentage of Calories from Groups of Subjects subjects number regimen Calories days Cal/day Protein Fat Carbohydrate A* Bt C* Dt Winter, 1957, Edinburgh Elt * This diet consisted of canned items in large variety with fresh bread. t This diet consisted of abundant amounts of fresh and frozen foods in wide variety. $ This diet consisted of dried milk solids with hydrogenated vegetable fat and carbohydrate added. Diet.-This is described summarily in Table I. The subjects were under continuous nutritional observation. It was intended that the amounts of the proximate principles of their diets should be representative of usual North American and British practice and should be adequate in quality. Unlimited fluids were provided. In the two American experiments the daily physical activity of all subjects was the same. Groups B and D received a luxury diet of the best procurable natural foods. Collection of Urine and Blood.-On the night before the observation food was last eaten at about 8 P.M. In the morning ml. of water was drunk and the subjects came, without food, to the laboratory. In the 1954 and 1955 experiments the subjects emptied their bladders and then lay down for 2 hr. without smoking, drinking or eating. During this period a specimen of urine, timed to the nearest minute, was collected and its volume measured to the nearest ml. Venous blood was drawn at the midpoint. In the 1957 experiments the subjects were ambulatory and no blood was drawn. Quantitative Analysis.-There is as yet no rapid and accurate quantitative method for determining total ketone bodies. In particular, the oxidation of betahydroxybutyric acid to acetoacetic acid by dichromate at 100 C. has usually been reported as only per cent complete. We have succeeded in making this yield 100 per cent by using temperatures over 1000 C. together with metaphosphoric acid, which catalyzes the oxidation. This substance has the further advantage of being an excellent protein precipitant.

3 Ketones in Serum and Urine For total ketone bodies, 10 ml. of 0 09 M-metaphosphoric acid are placed in a cone-tipped centrifuge tube. To this is added 0 5 ml. of serum or urine, and the whole is mixed by inversion. After centrifuging, 3 ml. of the supernatant are placed in an ampoule (or, rather better, in a screw-topped tube fitted with a teflon plug) and then 1 ml. of 3 N-sulphuric acid containing potassium dichromate in 0-02 M concentration is added. The ampoule or tube is sealed and autoclaved for 1 hr. at 15 lb. pressure. After cooling, the mixture is analyzed by the method of Michaels, Morgan, Liebert and Kinsell [1951] or by the method of Thin and Robertson [1952]. The first relies on the formation of the 2: 4 dinitrophenylhydrazone of acetone, which is extracted into carbon tetrachloride; in the second acetone diffuses in a Conway plate into alkaline salicylaldehyde. In both methods the intensity of the final colour is measured. Reagent blanks and standards of calcium zinc betahydroxybutyrate are included in each batch of samples. Qualitative Analysi8.-The method of Rothera [1908] has been standardized to give maximum sensitivity and uniform comparability from time to time. About 5 g. of anhydrous ammonium sulphate is added to a clean dry test tube; there must be enough to saturate the final solution and leave an undissolved excess. In succession and with mixing after successive additions, 0.5 ml. of urine, 0-2 ml. of freshly prepared 0 07 M-sodium nitroprusside, and 0-2 ml. of 5-N-ammonium hydroxide are added. Colour is recorded as 0-4 plus at 2, 5 and 10 min., and the maximum reading is accepted. RESULTS Quantitative: Serum.-Between winter and summer there were no significant differences in concentrations of total ketone bodies in the serum (Table II), and no significant differences between groups A and C and groups TABLE II.-CONCENTRATIONS OF TOTAL KETONE BODIES IN SERUM Group of Observations Mean Standard Coefficient Outdoor subjects number m-mole/l. deviation of variation temperature m-mole/l. per cent C. A to +27 B to +27 C to +7 D to Weighted means, Indiana plus Wisconsin AC BD all together X21 30 Statistical analysis: by students' "t" test, no differences between groups or seasons were significant at a probability level of 5 per cent. B and D. Therefore, a weighted mean for all subjects is justified. For all 198 specimens the mean was 0-71 m-mole/l., the standard deviation 0-21 m-mole/l. and the coefficient of variation 30 per cent. Quantitative: Urine.-The urinary excretion of total ketone bodies varied significantly with season (Table III). In severe winter cold excretion was greatest; in warm summer weather, least; and in the moderate winter

4 342 Johnson, Sargent and Passmore weather of Scotland, intermediate. In all groups, the coefficient of variation was larger than for serum. No weighted means would be statistically permissible for urine. TABLE III.-URINARY EXCRETION OF TOTAL KETONE BODIES Standard Coefficient Outdoor Groupjes o O bservationsemean deviation of variation temperature sm-mole/min. per cent 0 C. A to +27 B 11 1O to +27 C to +7 D * to +7 Winter, 1957, Edinburgh E to +14 Statistical analysis: by students' "t" test, differences were not significant between groups within summer and within winter at the 5 per cent level. All differences between A and B groups and C and D groups were significant at the 1 per cent level or below. Differences between the E groups and all AB and CD groups were significant at the 5 per cent level or below. TABLE IV.-ROTHERA REACTION IN URINE Observations Strength of reaction Groups nubservaln Groups number 0 tr A B C D Winter, 1957, Edinburgh E Statistical analysis: by the "Chi Square" test, there was no difference between A and C or B and D groups within summer or within winter, significant at the 5 per cent level. There was a difference between both winter 1954 groups, and all summer 1955 and winter 1957 groups, significant at the 0-01 per cent level. Qualitative: Urine.-There was a significant difference between severe winter weather and summer or mild winter weather (Table IV). Among the 394 specimens tested, positive reactions of + 1 or above were obtained only in Wisconsin. These qualitative findings for acetone plus acetoacetic acid are in agreement with the quantitative results of Table III for total ketone bodies; and they confirm the observations of Sargent and Consolazio [1950] for conditions of extreme cold.

5 Ketones in Serum and Urine 343 DISCUSSION The interpretation of means and ranges is always a matter of statistical judgment. For total ketone bodies in serum, we suggest that twice the standard deviation be considered the normal range. Our groups B and D had the higher mean figure. If this is taken and two standard deviations added, the upper limit of normal for total ketone bodies in serum is 1-4 m-mole/l., regardless of season. The existence of a correlation with temperature complicates the statistics for urinary excretion. However, as a general rule we should place 5,u-mole/min. as the upper limit of normal, realizing that it will be less in warm weather. Recalculation of data in the Handbook of Biological Data [Table 116, Albritton, 1953] gave an average of 2-3 ju-mole/min., and an upper limit of 5 7, for daily specimens collected without control of diet or environment. For physiological studies of ketone metabolism we urge the use of units of chemical equivalence, reform of inaccurate terminology, and standardization of conditions for collecting urine and blood. It has been common for investigators to express their results in terms of mg. of acetone per 100 ml. of urine or blood. This can lead to confused thinking. Table V demonstrates that, if one considers only concentration by weight in the blood, glucose would appear to be the predominant carbohydrate TABLE V.-SOME ILLUSTRATIVE CONCENTRATIONS OF INTERMEDIARY METABOLITES IN BLOOD Metabolite Conditions By weight By chemical Metabolite Conditions mg./100 ml. equivalence m-mole/l. Glucose... At rest Free fatty acid.. At rest Lactic acid.. At rest Lactic acid.. After exercise Pyruvic acid.. At rest Pyruvic acid.. In severe beriberi Ketone bodies.. At rest In nutritional ketosis (or derivative), and total fatty acid the predominant fat (or derivative). As judged by equivalent chemical units, however, such is certainly not always the case. In nutritional ketosis the molar concentration of total ketone bodies may equal that of glucose and far exceed that of free fatty acids; and after anaerobic work of short duration, the molar concentration of lactic acid is often at least double that of glucose. Even metabolites that are normally present in very small concentrations, like pyruvic acid, under some circumstances may become quantitatively important. Terminology for discussions of ketosis needs modification. The terms "ketonuria " and "ketonaemia" were originally given a pathological significance, because they were based on qualitative tests which could not detect small concentrations. However, modern quantitative methods show that there are always measurable concentrations of ketone bodies in urine and

6 344 Johnson, Sargent and Passmore blood. Therefore, the pathological connotation might better be served by the terms "hyperketonuria" and "hyperketonsemia". We recommend strongly the routine use of timed specimens of urine collected when the subjects are post-absorptive, and blood drawn during the same period. The two advantages that would accrue would be comparability of data from laboratory to laboratory, and applicability of data to calculation of renal parameters. In view of the correlation between temperature and renal excretion of total ketone bodies, we recommend control of the environment, or, if that is not possible, recording of environmental conditions under which the study may be conducted. ACKNOWLEDGMENTS All quantitative analyses were performed by Mrs. Evelyn Robbins, Mrs. Laura Sawyer and Mr. David Shirling, whose skill and industry we acknowledge here. Qualitative analysis was done by R. E. Johnson and N. Sperelakis. This investigation was supported in part by contract AF 18(600)-80 between the U.S. Air Force and the University of Illinois; and in part under a grant to R. Passmore from the Medical Research Council. In R. E. Johnson was on sabbatical leave and the recipient of a U.S. National Science Foundation Senior Post-doctoral Research Fellowship for study at Edinburgh University. F. Sargent, II, was on sabbatical leave and the recipient of a John Simon Guggenheim Memorial Foundation Fellowship for study at Oxford University. REFERENCES ALBRITTON, H. (editor) (1953). Handbook of Biological Data. Washington, D.C.: National Research Council. MICHAELS, G. D., MORGAN, S., LIEBERT, G. and KINSELL, L. W. (1951). "Studies in fat metabolism. I. The colorimetric determination of ketone bodies in biological fluids", J. clin. Invest. 30, ROTHERA, A. C. H. (1908). "Note on the sodium nitroprusside reaction for acetone", J. Physiol. 37, SARGENT, F., II and CONSOLAZIO, C. F. (1951). "Stress and ketone body metabolism", Science, 113, THIN, C. and ROBERTSON, A. (1952). "The estimation of acetone bodies", Biochem. J. 51,