FATTY-ACID COMPOSITION OF LIPID FRACTIONS IN DIABETIC SERUM

FATTY-ACID COMPOSITION OF LIPID FRACTIONS IN DIABETIC SERUM

285 The quality of this teaching compares favourably with that of any other country, but its quantity leaves much to be desired. The problem of supply...

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285 The quality of this teaching compares favourably with that of any other country, but its quantity leaves much to be desired. The problem of supply and demand is difficult. Until we can create the demand for trained doctors in industry, it is unlikely that the profession will come forward in sufficient numbers for training in this

specialty. Research As befits the inheritors of the tradition of Italy is one of the leaders in research.

Ramazzini,

The Clinica del Lavoro in Milan was founded by Luigi Devoto over fifty years ago. Today, under the leadership of Enrico Vigliani, it is as prolific as ever. The 20 institutes of E.N.P.I. throughout the country, though primarily in the service of industry, inevitably turn up research projects. The work of the institute at Helsinki in Finland is well known, and there are also the institutes in Zagreb and Prague, and the eleven in the Soviet Union. In the United States the Mellon Institute in Pittsburgh and the Kettering Institute in Cincinnati have many visitors from this country. A new occupational health institute has lately been set up within the High Institute of Public Health in Alexandria in Egypt. In this country the Medical Research Council has an Industrial Medicine Research Unit at the London Hospital under Donald Hunter-shortly, alas, to disappear. The contributions of its Pneumoconiosis Research Unit at Cardiff, its Toxicology Research Unit at Carshalton, and its Social Medicine Research Unit at the London Hospital certainly need no apologies. The industrial research associations, such as those for the cotton industry, the ceramic industry, the iron and steel industry, and the boot and shoe industry, have made notable contributions to dust-control and ergonomics.

The calibre of the research in this country is as high as anywhere else in the world, if not higher; but there is still not enough of it. If we develop our industrial hygiene services, new research projects will appear and will call for better research facilities. Conditions of Service In most countries the doctors are

paid by the industry.

employed by industry Exceptions are Bulgaria,

Czechoslovakia, and the Soviet Union. In Germany, France, and Scandinavia, the hiring and firing and conditions of service of industrial medical officers depends In on an agreement between the employers and the workers. Germany the national association of industrial medical officers is also a signatory to the agreement. The doctor cannot be dismissed without the concurrence of the works safety committee composed of representatives of employers and workers, and in Germany and France without the approval of the medical inspector of factories of the area. Conclusions If we could have the legislative arrangements of France, the medical examination scheme of Bulgaria, the chain of institutes of Italy, the agreements of Germany and Scandinavia, the teaching arrangements of the U.S.S.R., Finland, and Yugoslavia, the blanket coverage compensation scheme of Canada, the instrumentation of Czechoslovakia, and the enormous voluntary effort of the United States linked to our own Factory Inspectorate which is without doubt the finest in the world, we should indeed be paragons of occupational health. But we should not be British. As it is, we have the National Health Service, with all its faults; the National Insurance scheme, with its unevenness; the rehabilitation units; a few group services; the good will of a number of enlightened employers; the Royal Society for the Prevention of Accidents; a handful

teaching centres, two or three occupational hygiene services; the appointed factory doctors; the pneumoconiosis panels; our Factory Inspectorate; and a lot of muddled thinking. This is the British picture. Is it so of

bad ? With all the sincerity of the prodigal son I can assure you that Britain is still in the forefront-but only just. Great things are afoot in the world, and if we are not to be left behind, we must improve our industrial hygiene services, and expand and coordinate our system of medical examinations in such a way as to deal with all the problems of occupational health and safety in every factory, small as well as large. This demands a service which cannot be left exclusively to the voluntary effort of employers, though many of them have shown the way. Though we can still stand up to comparison with any other country in the world, we must put our house in order against the future. REFERENCES

Joint ILO/WHO Committee on Occupational Health (1957) Occup. Safety Hlth, 7, 113. Lancet (1962) ii, 1233. Shoib, M. O. (1959) J. Egypt. Publ. Hlth Ass. 34, 112.

FATTY-ACID COMPOSITION OF LIPID FRACTIONS IN DIABETIC SERUM E. BOEHLE

W. SCHRADE* M.D. Bonn

M.D. Frankfurt

PROFESSOR OF MEDICINE

CLINICAL ASSISTANT

R. BIEGLER

E. HARMUTH

M.D. Frankfurt

Ph.D. Ithaca

CLINICAL ASSISTANT

RESEARCH ASSISTANT

From the First Medical Clinic,

University of Frankfurt, Germany

INVESTIGATIONS of blood-lipids in human diabetes have hitherto been mainly concerned with cholesterol and the phospholipids. More recently, attention has been paid to the triglycerides (Pomeranze et al. 1950, Albrink et al. 1958, Aldersberg et al. 1959, Eggstein 1960, Finley 1961) and the nonesterified fatty acids (Bierman et al. 1957). But no systematic investigation of the fatty-acid composition of the different serum-lipids in diabetes has been published, although Hallgren et al. (1960) have analysed the fatty acids in the blood of 7 diabetics by gas-liquid

chromatography. We have therefore determined the levels of total lipids, cholesterol esters, free cholesterol, phospholipids, triglycerides, and nonesterified fatty acids, as well as the fatty-acid composition of these fractions, in the serum of forty healthy subjects and in that of a hundred and eighteen diabetics of different types and controlled with varying success. Our results confirm those of earlier studies of smaller numbers of subjects by spectrophotometric and gas-chromatographic methods (Schrade et al.

1958, 1959, 1960a). Patients and Methods Only male patients were included in this study. They received the usual hospital diet for diabetics. No additional fats rich in highly unsaturated fatty acids were given. The control subjects were healthy men free from any metabolic disturbance. They were divided into two groups: group C i comprised twenty men aged from 18 to 40 years (mean 29), and group C II contained another twenty between 45 and 67 years

(mean 55). The patients with diabetes mellitus

were

also divided into

groups. * Professor Schrade died

on

Sept. 29,

1962.

286 TABLE I-SERUM-LIPID CONCENTRATIONS IN NORMAL AND DIABETIC

Well-controlled Diabetes Mean fasting blood-sugar below 130 mg. per 100 ml. Group Ia.-Twelve juvenile diabetics. Age 16-30 years (mean 23). Duration of diabetes 1-7 years (mean 3.7). All cases treated with insulin (28-64) units (mean 37). No evidence of Kimmelstiel-Wilson syndrome, retinopathy, or atherosclerosis. Group Tie.—Twenty-seven elderly diabetics. (i) Twelve without hyperlipidsemia. Age 52-67 years (mean 59). Duration of diabetes 0-5-8-0 years (mean 3-9). Five were being treated with insulin (16-32 units (mean 22), four with tolbutamide, and three with diet alone. No evidence of Kimmelstiel-Wilson syndrome or retinopathy. One patient had a history of coronary infarction, one had sclerosis of the cerebral arteries, and two had sclerosis of peripheral arteries. (ii) Fifteen with hyperlipidaemia. Age 48-67 years (mean 55). Duration of diabetes 0-5-12-0 years (mean 4-1). Seven treated with insulin 16-40 units (mean 26), five with tolbutamide, and three with diet alone. No evidence of Kimmelstiel-Wilson syndrome or retinopathy. Ten patients had a history of coronary infarction, and three had sclerosis of the peripheral arteries.

Insufficiently Controlled Diabetes Twenty-five juvenile diabetics. Group Ib.-Ten with fasting blood-sugar of 156-248 mg. per 100 ml. (mean 209). Age 17-34 years (mean 26). Duration of diabetes 1-8 years (mean 4’9). All treated with insulin 48-72 units (mean 62). No evidence of Kimmelstiel-Wilson syndrome, retinopathy, or atherosclerosis. Group Ic.-Nine with fasting blood-sugar of 308-460 mg. per 100 ml. (mean 414). Age 19-34 years (mean 27). Duration of diabetes 1-7 years (mean 4). All treated with insulin 36-64 units (mean 52). No evidence of Kimmelstiel-Wilson syndrome retinopathy, or atherosclerosis. Group Id.-Six, in diabetic coma, with blood-sugar of 720-1070 mg. per 100 ml. (mean 931). Age 20-52 years (mean 36). Duration of diabetes 2-30 years (mean 152). Twenty-three elderly diabetics. Group IIb.-Ten with fasting blood-sugar of 154-218 mg. per 100 ml. (mean 186). Age 50-77 years (mean 59). Duration of diabetes 1-10 years (mean 4-3). Five treated with insulin 12-36 units (mean 22), four with tolbutamide, and one with diet alone. One had a history of coronary infarction, two had sclerosis of the cerebral arteries, and three had sclerosis of the peripheral arteries. None had evidence of KimmelstielWilson syndrome or retinopathy.

SUBJECTS

Group IIc.-Eight with fasting blood-sugar of 276-444 mg, (mean 364). Age 55-72 years (mean 62). Duration of diabetes 2-12 years (mean 7°6). Seven treated with insulin 16-40 units (mean 28) and one with diet alone. One patient had a history of coronary infarction and two had sclerosis of the per 100 ml.

peripheral arteries. Group IId.-Five, in diabetic coma, with fasting bloodsugar of 678-952 mg. per 100 ml. (mean 826). Age 56-71 years (mean 62). Duration of diabetes 4-10 years (mean 6-4). Diabetes with Kimmelstiel- Wilson Syndrome Group 777.—Nine diabetics with fasting blood-sugar of 118-210 mg. per 100 ml. (mean 157). Age 38-64 years (mean 51). Duration of diabetes 5-27 years (mean 15). All treated with insulin 24-72 units (mean 52). All had proteinuria, hypertension,and retinopathy; six had renal insufficiency with level of non-protein nitrogen in blood 35-4-126 mg, per 100 ml. (mean 61-2). Diabetes and

Idiopathic 77ype?-/!p:JfSM!M

Group IV.-Five diabetics with fasting blood-sugar of 117-160 mg. per 100 ml. (mean 137). Age 29-56 years (mean 43). Duration of diabetes 2-8 years (mean 5-2). All treated with insulin 20-64 units (mean 43). No evidence of Kimmelstiel-Wilson syndrome or retinopathy. One patient had a history of coronary infarction and one had sclerosis of the peripheral

arteries.

overnight fast, venous blood was taken from each subject for investigation of the serum-lipids. Total lipids were separated by extraction with a boiling mixture of ethanol and ether (3 vols.: 1 vol.) in the presence of 0-3 ml. of 0-% hydroquinone. Each 6 ml. of serum was subjected to three extractions -with 120 ml., 60 ml., and 60 ml. of the boiling mixture. The extract was then evaporated to dryness under nitrogen, reextracted with cold chloroform, filtered, and dried. Phospholipids were separated from the other lipids by dialysis through rubber (van Beers et al. 1958), and the non" esterified fatty acids were isolated withAmberlite IR 45’ (Biegler et al. 1960). Cholesterol esters, free cholesterol, and triglycerides were fractionated by chromatography ana silicic-acid column (12 g. Mallinkrodt 100 mesh) and eluted After

an

with 200 ml. of 1 % ether in hexane, 250 ml. of 5% etherin hexane, and 200 ml. of 25% methanol in ether. The amounts of cholesterol (Schonheimer and Sperry 1934 Sperry and Webb 1950), lipid phosphorus (Allen 1940), and esterified fatty acids (Antonis 1960) in the lipid fractions were

287

determined. Total lipids were calculated as the sum of lipid fractions derived from these determinations. The sum of fatty acids from cholesterol esters, phospholipids, triglycerides, and nonesterified fatty acids was designated " total fatty acids ". Fatty acids from the various fractions were methylated by refluxing for 90 minutes with methanol containing 2% (vol. per vol.) concentrated sulphuric acid after the addition of 0-1 ml. of 0-1% hydroquinone. The esters were analysed by gas-liquid chromatography on’Reoplex 400’ adsorbed on’Chromosorb W’ (80-100 mesh) as stationary phase (120 cm. column; apparatus of W. G. Pye, Cambridge). The t test was applied to check the significance of results; when p was less than 0-05, a difference was

regarded as significant. Results The Different Fractions In the juvenile diabetics, the values for total lipids and

total fatty acids were higher than in the controls (C i); and the poorer the control of the diabetes, the higher these values (table i). Of the different lipid fractions, triglycerides and nonesterified fatty acids were most increased. In the well-controlled diabetics (group la) and those with least insufficiently controlled diabetes (group ib), these fractions were the only ones increased. In the poorly controlled patients (group ic), the serum levels of cholesterol esters and phospholipids were also raised; but these rises were small compared with those in triglycerides and nonesterified fatty acids. In diabetics in coma (group id), the considerable increase in cholesterol esters and phospholipids was far exceeded by that in

triglycerides. In the elderly diabetics the results

TABLE II-CHOLESTEROL-ESTER FATTY ACIDS IN CONTROL AND DIABETIC

SUBJECTS

were

somewhat

288

Where the diabetes was well controlled, triglycerides and nonesterified fatty acids were increased (groupIIa (i)) but only the latter increase was significant. t Some elderly patients, however, with well-controlled diabetes, had a general hyperlipidaemia and all lipid fractions were significantly increased (group IIa (ii)). Likewise, in the elderly diabetics who were poorly controlled or in coma (group lib, c, and d), all lipid fractions were considerably increased. In all groups, the increase in triglycerides was more pronounced than that of cholesterol esters or phospholipids. The same was true in the diabetics with Kimmelstiel-Wilson syndrome (group III) and in those with idiopathic hyperlipidaemia (group iv). Fatty-acid Composition of the Various Fractions Cholesterol esters.-In those juvenile diabetics (groups Ia and b) with a normal level of cholesterol ester in the serum, the fatty-acid composition of this ester did not differ from that in the controls (table 11). In group ic, the small elevation in cholesterol ester was due to slight increases in the esters of palmitic, palmitoleic, stearic, oleic, and linoleic acids; the amounts of other fatty acids were not increased, and cholesterol arachidonate was even somewhat decreased. In the juvenile diabetics in coma (group id), all fatty acids in the cholesterol-ester fraction contributed to the extreme increase of cholesterol ester, though to different extents. As a result, the proportions of palmitoleic and oleic acids were significantly higher, and those of linoleic and arachidonic acids significantly lower than in the control subjects. Among the elderly diabetics, those with no increase in serum-cholesterol (group lia (i)) had slightly elevated levels of cholesterol palmitate and slightly depressed levels of linoleate and arachidonate. In groups IIa (ii), lib, and lie the pronounced increase in cholesterol ester was associated with large increases in the esters of all saturated fatty acids, somewhat slighter increases in the esters of palmitoleic acid, and only very small increases in those of oleic acid. The serum levels of dienoic-acid and tetraenoic-acid esters were not raised. Consequently, in these patients, the cholesterol ester contained a higher proportion of saturated acids and a lower proportion of unsaturated acids than in the control subjects (group C II). The pronounced elevation of cholesterol ester in the elderly patients in diabetic coma (group lid) reflected increases in fatty-acid esters of all types ; but the levels of saturated and monounsaturated acids rose much higher than those of polyunsaturated acids. This caused changes in the proportions of fatty acids in the cholesterol ester similar to those in the other groups: the proportion of palmitic acid was significantly higher and that of linoleic acid and arachidonic acid significantly lower than in the control subjects. Similarly, in patients with Kimmelstiel-Wilson syndrome (group III), the high serum level of cholesterol ester was due to uneven increase in esters of the different fatty acids. In patients with idiopathic hyperlipidaemia as well as diabetes (group iv), the elevation of the serum-cholesterol levels was largely a reflection of the greater amounts of cholesterol esters of the saturated fatty acids and palmitoleic acid; the amounts of oleic-acid, linoleic-acid, and arachidonic-acid esters were also increased but to a smaller extent. The esters of trienoic, pentaenoic and hexaenoic acids were distinctly increased. In consequence, the proportion of palmitic acid was significantly higher and- that of linoleic acid significantly lower than in the control subjects (group C II).

similar.



t Multigraphed copies of tables giving details of results may be had from the office of The Lancet, 7, Adam Street, London, W.C.2

well-controlled juvenile diabetics of phospholipid was normal, as level (group la), was the fatty-acid composition of the phospholipid. In the less well-controlled juvenile diabetics (groups ib and c), the serum-phospholipid level was slightly raised, together with the levels of all component fatty acids. The elevation of phospholipid level in the comatose patients (group id) was accompanied by non-uniform increase in the different fatty acids. The ’rise in saturated and monounsaturated fatty acids was more pronounced than that in dienoic and tetraenoic acids. Consequently, the relative proportions of the former were increased and those of the latter diminished. The increase in the proportion of palmitic acid and the decrease in that of arachidonic acid and linoleic acid were significant. In the well-controlled elderly diabetics of group lia(i), the fatty-acid composition of the phospholipid was normal. In those with hyperlipidaemia and in those whose diabetes was poorly con. trolled, serum-phospholipid was increased; the increase was largely due to a rise in palmitic acid, while amounts of other saturated and monounsaturated fatty acids were increased to a smaller extent. If, however, there were any increase in linoleic acid it was hardly noticeable, and arachidonic acid was not increased at all. In the extreme hyperphospholipidaemia which is part of the hyperlipidaemia of diabetic coma (group lid), the lack of uniformity between the increases in different fatty acids was still more pronounced. Uneven increases in phospholipid fatty acids were also found in the hyperphospholipidxmic patients with the Kimmelstiel-Wilson syndrome (group III) and in patients with idiopathic hyperlipidaemia as well as diabetes mellitus (group iv). In group III, the proportionate increase in palmitic acid was significant, as was the proportionate decline in arachidonic and linoleic acids; in group rv, only the decrease in the proportion of linoleic acid was significant.

Phospholipids.t-In the

serum

the juvenile diabetics, the rise level was not shared uniformly by in serum-triglyceride the different fatty acids. There was only a slight tendency for the saturated and monounsaturated fatty acids to show larger increases than polyunsaturated fatty acids. In the extreme elevation of triglycerides seen in diabetic coma, the inequality of the increases in triglyceride fatty acids was more pronounced. As a result, there were significant increases in the proportions of palmitic and palmitoleic acids and significant decreases in the proportions of linoleic and arachidonic acids in these patients. In the wellcontrolled elderly diabetics of group lla(i), in whom serum-triglyceride levels were not significantly raised, the proportion of oleic acid was significantly increased and that of arachidonic acid significantly diminished. In the other groups of elderly diabetics, where the rise in serum-triglycerides was great, amounts of saturated and monounsaturated fatty acids were usually increased more than those of dienoic and tetraenoic fatty acids. The elevation in triglyceride level in patients with the Kimmelstiel-Wilson syndrome (group III) was likewise associated with larger increases of saturated fatty acids and oleic acid than of polyunsaturated fatty acids. The differences, however, were only slight. In patients with idiopathic hyperlipidxmia as well as diabetes, serum-triglyceride levels were very high, and once again the rise in saturated fatty acids and oleic acid exceeded that in polyunsaturated fatty acids. But these changes in fatty-acid composition of the triglycerides were not quantitatively significant.

Triglycerides.t-Among

Nonesterified fatty acids. t-In

all the

juvenile diabetics

289

levels of all nonesterified fatty acids were elevated, although to varying degrees. This variation was most striking in diabetic coma when the elevation of nonesterified fatty acids is greatest (group id). The rise in polyunsaturated fatty acids was less pronounced than that in other fatty acids. Among these others, oleic acid was most increased. Similarly, in the elderly diabetics, levels of saturated and monounsaturated fatty acids were, in general, more elevated than those of the polyunsaturated fatty acids. In patients with Kimmelstiel-Wilson syndrome (group III) and in those with diabetes associated with idiopathic hyperlipidaemia, amounts of nonesterified saturated and monounsaturated fatty acids were increased more than those of the nonesterified polyunsaturated acids. Discussion The patients studied illustrate different types of serumlipid distribution in diabetes mellitus. In juvenile patients where the diabetes is well controlled, serum levels of cholesterol and phospholipid are normal whereas the levels of triglycerides and nonesterified fatty acids may be slightly elevated. In elderly patients, good diabetic control is not always associated with this characteristic distribution ’of serum-lipids. There may be increases in all the lipid fractions, a type of hyperlipidaemia which is found frequently in non-diabetic patients with atherosclerosis; and, in fact, all such diabetics in our group’ na(ii) showed evidence of atherosclerosis. Non-diabetic atherosclerotic patients may have no hyperlipidmmia (in our experience, approximately 25% of all atherosclerotic patients have normal serum-lipid values); and our study included several elderly diabetics without hyperlipidaemia (group na(i)) who nevertheless had evidence of atherosclerosis. Insufficient control of diabetes is associated with increased serum-lipid levels. Juvenile diabetics in particular react to slightly inadequate control with increases in triglycerides and nonesterified fatty acids; when the inadequacy is more pronounced, even to the point of ketosis, they react like elderly diabetics with increases in cholesterol and phospholipids as well. The KimmelstielWilson syndrome is associated with rises in all serum-lipid levels, even when the carbohydrate metabolism is well controlled, neither can good control of carbohydrate metabolism abolish the elevation of serum-lipid levels in patients with diabetes mellitus and idiopathic hyperlipidxmia. In each instance, the increase in triglycerides exceeds that in cholesterol and phospholipids, which suggests that triglycerides may have the more fundamental role. In the hyperlipidaemia of diabetes the relative proportions of the serum-lipids are changed. In the control subjects, for example, 32% of total lipids existed as cholesterol ester, 30% as phospholipid, and 23% as triglyceride, whereas, in juvenile diabetics in coma, only 17 % existed as cholesterol ester and 19% as phospholipid, but 53% as triglyceride. Moreover, in all groups, diabetic hyperlipidaemia was associated with changes in fatty-acid content which seemed to be qualitatively similar. In all lipid fractions, polyunsaturated fatty acids increased less than saturated and monounsaturated fatty acids, and this may reflect different turnover rates in the metabolism of

fatty acids. "

It is

probably relevant that polyenoic fatty

acids are essential " substances and not unlimitedly available. The changes in fatty-acid distribution in diabetic hyperhpidxmia are similar to those found in patients with atherosclerosis or idiopathic hyperlipidaemia (Schrade et al.

1961a) : elevation of serum-cholesterol, serum-phospholipid, and serum-triglyceride levels is again generally associated with greater increase of saturated and monounsaturated fatty acids than of polyunsaturated fatty acids in these fractions. According to our preliminary results, the same changes are also found in other symptomatic hyperlipidaemia such as myxoedema and the nephrotic syndrome (Schrade et al., 1961b). Apparently we are dealing here with a non-specific phenomenon. Whether the overloading of blood with esters of saturated and monounsaturated fatty acids is of special pathological significance-e.g., in the development of atherosclerosis-is not certain. The suggestion has been made that these esters have a greater tendency to be deposited in bloodvessels than esters of polyunsaturated fatty acids; but this has yet to be proved. Our investigations show that, in some ways, the lipid substances in blood provide a better measure of the quality of diabetic control than the blood-sugar. Some lipid fractions (triglycerides and nonesterified fatty acids) could be increased even when the fasting blood-sugar level was normal. The significance of nonesterified fatty acids in diabetes has been mentioned already by Bierman et al. (1957). Similarly, Werk et al. (1961) have shown that the ketone content of blood is a more sensitive measure of diabetic control than blood-sugar. As long as the levels of triglycerides, nonesterified fatty acids, and ketones are elevated in the fasting state, the metabolic disturbance of diabetes cannot be regarded as completely corrected. The control of diabetes cannot, however, be assessed by blood-lipid levels in patients in whom the complications of diabetes (e.g., Kimmelstiel-Wilson syndrome) or other coincident disturbance in lipid metabolism (e.g., " atherosclerotic " or idiopathic hyperlipidaemia themselves cause a rise in serum-lipids. The hyperlipidaemia then reflects more than one metabolic disturbance. Only that part which is due to insufficient control of diabetes can be eliminated by correction of the disturbance in carbohydrate metabolism; elimination of the other requires additional treatment-e.g., low-fat diet, or a diet supplemented with fats rich in polyunsaturated fatty acids, or drugs to lower serum-lipids. In well-controlled elderly diabetics, to distinguish between diabetic and " atherosclerotic " elevation of serum-lipids is often most difficult. In our experience, the hyperlipidaemia found in non-diabetic patients with atherosclerosis is, in general, not associated with a rise in blood levels of nonesterified fatty acids and ketones; indeed, these levels are frequently depressed (Schrade et al. " 1960b). Their elevation in so-called " well-controlled elderly diabetics with hyperlipidxmia can be regarded as evidence of insufficient correction of the diabetes, besides the " atherosclerotic " elevation of blood-lipids. If the control of the diabetes is improved, the levels of nonesterified fatty acids and ketones fall. Any residual hyperlipidaemia can then be better characterised.

Summary A hundred and eighteen male diabetics were divided into several groups according to age, the degree of control of carbohydrate metabolism, and the presence of complicating factors. Forty normal subjects were divided into two control groups according to age. Serum samples were taken from each in the fasting state. Total-lipid extracts were fractionated into cholesterol ester, free cholesterol, phospholipids, triglycerides, and nonesterified fatty acids. The fractions were deter-

290 mined quantitatively and their fatty-acid composition elucidated by gas-liquid chromatography. In well-controlled diabetes, serum levels of cholesterol and phospholipids were normal, whereas those of nonesterified fatty acids and triglycerides were raised. Some of the well-controlled elderly diabetics had increases in all lipid fractions due to the associated hyperlipidsemia characteristic of atherosclerosis. Where control was slightly inadequate, particularly in juvenile diabetics, levels of triglycerides and nonesterified fatty acids were alone raised. But where the inadequacy was pronounced and in diabetic coma all lipid fractions were increased-as they were in the Kimmelstiel-Wilson syndrome and in diabetes mellitus plus idiopathic hyperlipidoemia. Wherever there was an increase in total lipids, the increase in triglycerides was considerably larger than that in cholesterol ester and phospholipids. The elevation of blood-lipid fractions in diabetes was associated with increased amounts of fatty acids. But different fatty acids were increased to different extents. Esters of saturated and monounsaturated fatty acids were increased more than those of polyunsaturated fatty acids. Reduced amounts of arachidonic-acid esters were found in several groups. Owing to these disproportionate increases, the relative fatty-acid composition of the various fractions differed from that in the controls: the proportions of saturated and monounsaturated fatty acids were higher in all fractions, while those of polyunsaturated fatty acids were lower. We are grateful to the Deutsche Forschungsgemeinschaft for a REFERENCES

Adlersberg, D., Eisler, L. (1959) J. Amer. med. Ass. 170, 1261. Albrink, M. I., Man, E. B. (1958) Diabetes, 7, 194. Allen, R. J. L. (1940) Biochem. J. 30, 858. Antonis, A. (1960) J. Lipid Res. 1, 485. Biegler, R., Boehle, E., Schrade, W., Abt, W. (1960) Klin. Wschr. 38, 532. Bierman E. L., Dole, V. P., Robberts, T. N. (1957) Diabetes, 6, 475. Eggstein, M. (1960) Klinik der Gegenwart, München, 9, 593. Finley, J. K. (1961) Angiology, 12, 127. Hallgren, B., Stenhagen. S., Svanborg, A., Svennerholm, L. (1960) J. clin.

Invest. 39, 1424. Pomeranze, J., Kunkel, H. G. (1950) Proc. Amer. Diabetes Ass. 10, 217. Schönheimer, R., Sperry, W. M. (1934) J. biol. Chem. 106, 745. Schrade, W., Biegler, R., Boehle, E. (1958) Klin. Wschr. 36, 314. Boehle, E., Biegler, R. (1959) ibid. 37, 1101. Meder, V., Teicke, R. (1960a) ibid. 38, 126. (1960b) Lancet, ii, 1409. Biegler, R., Boehle, E. (1961a) J. Atheroscler. Res. 1, 47. — Boehle, E., Biegler, R. (1961b) Dtsch. med. Wschr. 86, 781. Sperry, W. M., Webb, M. (1950) J. biol. Chem. 187, 97. van Beers, G. J., de Jongh, H., Boldingh, J. (1958) in Essential Fatty Acids (edited by H. M. Sinclair); p. 43. London. Werk, E. E., Knowles, H. C. (1961) Diabetes, 10, 22. —











and suggests that similar changes take place in patients with hyperparathyroidism, and that such changes might be reversed by surgery. We describe here the results of an investigation into the urinary filterable non-dialysable carbohydrates before and after operation in patients with hyperparathyroidism. The observation that patients with kidney stones without hyperparathyroidism may have increased excretion of a urinary mucoprotein (Boyce and Swanson 1955) led us to include such a group in our work. Material and Methods

patients with hyperparathyroidism and bone disease (table i) and 8 patients with kidney stones without hyperparathyroidism (table n) were investigated. In all the patients with hyperparathyroidism a parathyroid adenoma was found and removed. The success of the operation was confirmed by complete reversion of the metabolic disturbances in all patients, 1 patient died eight days after operation. Urines were collected one week before the operation, and 8

grant in support of this work.



Fig. I-Carbohydrate fractions in 24-hr. urine of patients with kidney stones without hyperparathyroidism.



CARBOHYDRATES IN THE NON-DIALYSABLE FRACTIONS OF FILTERED URINE IN HYPERPARATHYROIDISM A. BORELLI

M. O. RIBEIRO LEITE

M.D. São Paulo

M.D. São Paulo

four weeks after surgery. two to

exclude the effect of

hour urine

specimens stored at 0°C withoutt the addition of were

preservatives, and

were

assayed immediately. The urines filtered

were

and dialysed for twentyfour hours

I. MARIANI

in ’Cello-

phane ’ tubing against tap 0°C. The contents of the dialysis

water at

D.M. São Paulo PROFESSOR AND HEAD OF DEPARTMENT

From the 1st Medical Clinic, Hospital das Clinicas, of São Paulo Medical School, Brazil

to

Twenty-four-

B.A. Sao Paulo

A. B. ULHÔA CINTRA

operation

University

DISSOLUTION of the bone-matrix, and kidney changes that suggest the tubular transport of mucoproteins, have been found in rats after injection of parathyroid extract (Engel 1952). The increased urinary excretion of mucoproteins in these laboratory animals (Engel and Catchpole 1953) seems to point to a relationship between parathyroid activity and the excretion of mucoprotein,

bag were brought to a

Fig. 2-Protein-free carbohydrates before and after operation in 24-hr. urines of patients with hyperparathyroidism.

known volume with distilled water, and used for chemical determinations (Melo et al. 1959). The modified anthmne method of Seifter and associates (Seifter et al. 1950) was used for the hexose determinations. Standards containing equimolecular amounts of galactose and mannose were run simultaneously with the unknowns. Serum-calcium (Clark and Collip 1925), serum-phosphorus (Fiske and Subbarow 19N. and serum-alkaline-phosphatase (Bodansky 1933) were deta-