Plasma glycerol and glycerol turnover during treatment of hyperthyroidism

CLINICA CHIMICA ACTA

PLASMA

GLYCEROL

TREATMENT

GUNNAR

189

AND GLYCEROL

TURNOVER

DURING

OF HYPERTHYROIDISM

TIBBLING,

EBBE

CEDERQUIST

AND ALF

LUNDQUIST

Departments of Clinical Chemistry, Radiotherapy and Internal Medicine, University Hospital, Lund (Sweden) (Received

February

3, 1969)

SUMMARY

The plasma glycerol concentration was studied in hyperthyroid patients during carbimazole treatment and after radioiodine therapy. The glycerol concentration varied generally in close correspondence to the change in protein-bound iodine, T, uptake test, and 1311uptake by the thyroid gland. The glycerol turnover rate decreased and was within normal limits when the treated patients were euthyroid. The results indicate that, besides the disappearance of the signs and symptoms of hyperthyroidism, the fat mobilization was normalized on adequate treatment; also that the determination of the glycerol concentration in blood provides information similar to that of conventional laboratory tests wl!en examining for hyperthyroid function.

Evidence has been given of increased fat mobilization and enhanced utilization of free fatty acids (FFA) in the hyperthyroid statel-s. The degree of fat mobilization is reflected by the glycerol level in bloodsyg. Two previous studies have shown that the determination of the blood glycerol, in contrast to the plasma FFA, enabled euthyroid and hyperthyroid subjects to be distinguished with only a slight degree of overlap?,*. In the present investigation, the variation of the glycerol concentration was studied in subjects with spontaneous hyperthyroidism during carbimazole treatment and after radioiodine therapy. The glycerol turnover was studied in a small group before and during successful treatment with carbimazole. MATERIALS

AND METHODS

Carbirnazole therapy g~oz$ This contained eleven hyperthyroid women and two hyperthyroid men (aged rg -62 years, mean 44 years), examined at the outpatient medical clinic. Hyperthyroidism was diagnosed according to the subject’s clinical symptoms, laboratory data, and response to treatment. The laboratory data consisted of basal metabolic rate, proteinbound iodine (PBI), [lz51]T3 uptake test (T3 test), and IS11uptake by the thyroid gland at 2 h and 24 h. They were examined on 95 occasions by one physician who, at the Clin. Chim. Acta, 25 (1969) rag-198

examinations, was unaware of the actual laboratory data. The mean observation period for the patients was 4.6 months (range 2-7 months). At each examination, PBI, T, test, and plasma glycerol were analysed on blood samples collected in the morning when the patients had been fasting for m-15 h. The glycerol turnover was studied in ten patients, eight women and two men, before and during adequate treatment with carbimazole.

This group consisted of IIZ untreated and radioiodine-treated hyperthyroid patients, 104 women and 8 men (aged. 38-86 years, mean 62 years), studied on 270 occasions at the radiotherapy department: 62 patients were studied before therapy and IOO after. Thirty-nine patients were studied only once: the rest had an average of 3.1 examinations. At each examination, 1311uptake by the thyroid gland at 2 h and 24 h was measured and PBI, T:, test, and plasma glycerol were analysed. The patients were examined at varying intervals from three months up to about two years after treatment. Patients on antithyroid drugs before and after treatment with radioiodine were excluded. Twenty-eight patients were retreated with radioiodine because of inadequate response to earlier therapy. The calculated dose to the thyroid gland was usually 104 rad. at the first treatment. The diagnosis of hyperthyroidism of the untreated patients was made according to the clinical symptoms and laboratory data. The treated patients were classified as hyperthyroid, euthyroid, and h3~othyroid, according to their clinical symptoms, by two physicians, one of whom was aware of the results of the Is11 uptake by the thyroid gland. Where the physicians disagreed in the classification of the subjects, the decision of the one who knew the results of the 13x1uptake was accepted. When admitted to the outpatient clinic, the patients were instructed to fast overnight. In the later part of the study, however, it became apparent that some had eaten a light morning meal two to three hours before the blood sa.mples were drawn. It cannot therefore be excluded that in 10-2074 of the examinations the patients were not completely fasting. In the study, diabetic patients were excluded, as diabetes by itself may be associated with an elevated glycerol level; patients on regular treatment with various drugs that interfere with sympathetic nervous activity were also excluded, since such drugs influence lipolysis to varying extent.

The subjects were allowed a quiet period of at least 15 min in sitting position before blood was collected from a superficial cubital vein. T3re blood samples for determining the plasma glycerol were kept on ice before separation of the plasma, usually within two hours. The plasma was then frozen until anal.ysed. Serum was used for PBI and T, test. Capillary blood samples were collected for determination of the glycerol turnover. The basal blood glycerol concentration was determined on samples taken after 30 min with the patient resting in supine position.

The plasma or blood glycerol was analysed by an enzymatic fluorometric C&n.Chim.

Acta,

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PLASMA GLYCEROL IN HYPERTHYROIDISM

micromethodlo.

The serum PBI was determined

191

by the use of the Technicon

Analyzer. The T, test was performed by determining the [lZ51] T, uptake dexll. The 1311uptake by the thyroid gland was determined according to mendations of the IAEA consultants group l2. The glycerol turnover was by continuous infusion of glycerol at constant rate according to a method describeda.

Auto-

by Sephathe recomdetermined previously

Normal values The normal ranges (95% confidence limits) for the laboratory parameters included in the study are given in Table I. For practical reasons, the glycerol deter-

TABLE

I

NoRlMALRANGESFOR THE LABORATORYPARAMETERS LaSwatory paranwters Glycerol plasma, venous (sitting position for 15 min) blood, capillary (supine position for 30 min) fractional turnover rate turnover rate Serum PBI T, uptake test I311 uptake by thyroid gland zh 24 h

Limits fou * 2 S.D. (Mean)

o.ozS-0.128(0.078)

mM

o.ors-0.078(0.048)

mM

o,oIg-0.055(0.037) min-l 0.45-1.65(1.05) ,umoles/min/kg 4.5-7.3(5.9) &Ioo ml* 8~120(100)~~* IO-35o/o**

zo-55%**

* Normal limits at the department of clinical chemistry. ** Normal range used at the department of radiotherapy.

minations were performed on venous plasma instead of capillary whole blood. The 95% confidence limits for the capillary blood concentrations obtained from 75 normothyroid subjects studied in sitting position after at least 15 min of rest are 0.0230.107 mM?. The glycerol concentration in whole blood is about 11% lower than in plasma*;. Furthermore, the mean capillary-venous difference in the resting forearm has been estimated at 8% (a = rg). The normal range calculated from the capillary blood glycerol concentrations was thus increased by 20% to be applicable to venous plasma concentrations. The normal limits for the capillary blood glycerol concentration, fractional turnover rate, and turnover rate in subjects resting in supine position have been calculated from the results obtained in 26 normothyroid subjects, mainly presented in a previous report*. RESULTS

Carbirnazole therapy groq3 The mean fasting plasma level of glycerol in eleven of the hyperthyroid patients was 0.176 mM (range 0.116-0.245 mM) before treatment. Of the remaining two patients, Clin.

Chim.

Acta,

25 (1969)

x89-198

TIBBLING

192

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1

2

Case 5

0

1

2

3

4

5

6

7

0

1

2

3

4

5months

4

I 5 nionths

Case 6

0

1

2

3

Fig. 1. Variation of plasma glycerol (0). PBI (a), and T, uptake test (L) in six hyperthyroid patients during treatment with carbimazole. The results are given in standard deviations (S.D.). The classification of the patient at each examination according to the clinical symptoms, independent of the actual laboratory data, are: H (hyperthyroid), E (euthyroid), M (hypothyroid) ; when in parentheses, the classification was uncertain. The carbimazole dose (mg/day) during the treatment is shown in the figure. The patient in case 6 had a moderate obesity.

one with a moderate obesity had a glycerol concentration of 0.342 mM, and the other with 0.187 mM was only studied in supine position after 30 min rest. The serum PBI concentration of the group averaged 13.4,~g per IOO ml (range 8.5-19.0 lug per IOO ml) and the T, test 167% (range 123-zoo~~). All patients responded well to the carbimazole treatment and became clinically euthyroid within one to two months. The variation of the plasma glycerol, PBI, and T3 test during the treatment is shown in Fig. I for six patients of the group. The cases I, z, 3, and 4 are representative for the group in the behaviour of these laboratory parameters. The parameters varied as a rule in a close correspondence and were generally within normal limits when the patients were judged to be euthyroid. Two patients, one of whom (case 4) is shown, developed slight to moderate symptoms of hyperthyroidism when the drug dose was reduced, but returned to euthyroid when the dose was again increased. In one woman (case 3) the levels of the PBI and T3 test returned to increased values when the carbimazole dose was reduced, and l-thyroxine was added because the patient’s clinical signs and symptoms indicated a slight overtreatment. Here, the glycerol concentration was within normal limits, in contrast to the increased levels of the PBI and T, test, when the patient was judged to be euthyroid. One man (case 5) with a glycerol concentration of 0.116 mM before treatment and one woman with 0.132 mM had very slight symptoms of hyperthyroidism. One Clin.

Chiw.

Acta,

25 (1969)

189-198

PLASMA GLYCEROL IN HYPERTHYROIDISM

I93

C!j.ceroi 1mM

0.300 I

0.010 0.020

0

0.030

0.040

0.050 0.060 0.070 min-’ Fract. turnover rate

2. Relationship between capillary blood glycerol concentration and fractional turnover rate of glycerol for the hyperthyroid patients studied before and during successful treatment with carbimazole (connected symbols) together with the results of hyperthyroid (e 9 ; q 6) and normothyroid (o 9; q $) subjects mainly presented in a previous report*. Horizontal and vertical lines correspond to normal limits for & 2 S.D. I refers to a woman with moderate obesity. Fig.

Group

IU

.

Group II Euthyroid Hyperthyroid

Group O-I Euthyroid Hyperand thyroid hypothyroid

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t t

m

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Fig. 3. Frequency diagram for the venous plasma glycerol concentrations obtained in the radioiodine therapy group (270 observations). Group III, Group II, and Group o-1 represent increased values in three, two, and none or one of the three laboratory tests: PBI, T, uptake test, and Ial1 uptake by the thyroid gland. The patients were classified in hyperthyroid, euthyroid, and hypothyroid according to the clinical symptoms. Symbols represent untreated (m), treated (o), treated patients where the classification was uncertain (A), and hypothyroid patient ( x ). Clin. Chim. Acta,

25 (1969) 189-198

TIBBLING ei! al.

194

hyperthyroid woman with moderate obesity (case 6) did not normalize her glycerol concentration on treatment, although she became clinically and laboratorily euthyroid. Nine patients of the group studied in respect of the glycerol turnover had a mean blood glycerol concentration of 0.104 mM (range 0.076-0.154) in the hyperthyroid state and 0.051 mM (0.035-0.068) in the euthyroid state during the carbimazole treatment. The corresponding mean values with the ranges for the fractional turnover and 0.038 min-I (o.ozg-o.o45), and for the turnrate were 0.049 min-l (o.o3g-0.066) over rate 3.09 pmoles/min/kg (2.30-4.82) and 1.18 pmoles/min/kg (0.71-1.57). The remaining patient was the obese woman who had the respective values of 0.302 mM, when hyperthyroid and 0.180 mM, 0.027 0.035 min-l and 5.78 pmoles/min/kg min.-l and 2.69 pmoles/min/kg when euthyroid. In one patient, the glycerol turnover was determined twice in the euthyroid state with an interval of two months. The drug dose was not changed during this period. The difference in the results between the two determinations for the blood glycerol concentration, fractional turnover rate, and turnover rate was about 5%. The correlation between the fractional turnover rate and the blood glycerol concentration for the patients before and during adequate treatment with carbimazole are shown in Fig. 2, together with the results obtained with hyperthyroid and normothyroid subjects, mainly presented in a previous reports. All the patients, except the obese woman, had results when they were euthyroid that agreed with those of the normothyroid subjects. Radioiodine therapy group The plasma glycerol concentrations for the patients studied are shown in a frequency diagram in Fig. 3, The results have been divided into three parts (Group III, Group II, and Group o-l) with regard to increased levels in three, two, and none or one of the three laboratory parameters : PBI, T, test, and la11 uptake by the thyroid gland. The sign for each examination refers to the classification of the patient according to the clinical symptoms in untreated hyperthyroid, and treated hyperthyroid, euthyroid, and hypothyroid. No separation has been made according to the occurrence of heavy, moderate, or slight symptoms of hyperthyroidism. In some treated patients, the classification in euthyroid or hyperthyroid has been questioned by the examining physicians because occurring symptoms could be caused either by a remaining hyperthyroid function or by some interfering disease, mainly cardiac fibrillation. These cases are shown in the figure. In a few instances, the laboratory tests gave evidence of hypothyroid function, although the patients were judged to be euthyroid, Since the glycerol concentration in blood does not enable us to distinguish between hypothyroid and normothyroid conditions’, they have not been signed separately. From the figure, the median glycerol concentration can be seen to decrease with increasing number of normal values for the laboratory tests. The variation of the plasma glycerol, PBI, T, test, and 1311uptake by thyroid gland after therapy is shown for three patients in Fig. 4. Case A is representative for the patients who responded adequately to therapy. It shows that the plasma glycerol, PBI, and T3 test varied in a close correspondence. In a few of these patients, however, the glycerol concentration was normalized later than the other laboratory parameters. Case B demonstrates the behaviour of the glycerol concentration in a patient who beClin. Chirn. Acta, 25

(1969)

189-198

PLASMA GLYCEROL

IN ~YPERT~YROI~IS~

“95

Case B

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12 months

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C

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21 months

plasma glycerol (0) , PBI (&), T, uptake test in), and Ia11 uptake by the thyroid gland at 2 h (----) and 24 h (-) for three typical cases in the radioiodine therapy group. The results for glycerol, PRI, and T, test are given in standard deviations (S.D.). For Is11 uptake, the vertical bars represent the respective normal range. The classification of the patients at each examination according to the clinical symptoms are: H (hyperthyroid), E (euthyroid), M (hypothyroid) ; when in parentheses, the classification was uncertain. Arrow indicates radioiodine treatment and close to it the amount of activity given. Fig.

4.. Variation

of

came hypothyroid during the observation period. In contrast to the decreased levels of the other laboratory tests, the glycerol concentration was normal in the hypothyroid state. Case C was a hyperthyroid woman with cardiac fibrillation. This case is representative for the patients who failed to respond to one treatment of radioiodine. This case shows again the simultaneous variation in the laboratory parameters. DISCUSSION

As recently demonstrated, most hyperthyroid patients can be distinguished from euthyroid subjects by determining the glycerol concentration in blood7p8. The plasma glycerol concentration was increased above the normal level in all except three of the untreated h~erth~oid patients studied here. One of these three, a man in the carbimazole therapy group, had a concentration of 0.116 ml@. This value, C&z. Chirn.

Acta,

25 (1969)

18g-rg8

196

however, is slightly increased when account is taken of the normal range for men only, which is calculated at 0.022-0.103 mM for venous plasma’. The corresponding range for women is 0.036-0.032 mM. Thus about 3% of the untreated hyperthyroid patients had a normal glycerol concentration; this is in agreement with the previous results?. The ordinary behaviour of the plasma glycerol, PBI, and T3 test (and 131Iuptake by thyroid gland for the radioiodine therapy group) in the patients when treated either with carbimazole or radioiodine was a simultaneous decrease in the laboratory parameters, being generally within normal limits when the patients were judged to be euthyroid. The simult~eous variation in the parameters was further evident in the patients who, after being euthyroid on treatment, returned to hyperthyroid when the carbimazole dose was reduced (Fig. I, case 4) or when the radioiodine therapy was unsuccessful (Fig. 4, case C). In these cases, all the values increased until the patient responded to further treatment. This relation between the laboratory parameters is also apparent in the frequency diagram in Fig. 3, where the combined results of the laboratory tests are shown fos the radioiodine therapy group. In the radioiodine therapy group, the patients classified as euthyroid after therapy are heterogeneous in respect to their thyroid function. Some of them were laboratory hypothyroid, although they had no clinical evidence of hypothyroidism. The opposite relation, patients with a slight remaining hyperthyroid function, can also be expected in this group. These patients may have a glycerol concentration at the upper normal level. This is evident in the four patients in Group III (all the three laboratory tests increased; Fig. 3) who were judged to be euthyroid with a glycerol concentration between O.IIO-0.139 mM. The laboratory data, however, might indicate a remaining hyperthyroid function in these patients, The main part of the group classified as hyperthyroid after therapy had increased glycerol concentrations. Not all the patients in the radioiodine therapy group were completely fasting at the examination, which could decrease the fat mobilization and result in a lower glycerol level than in the fasting state13. This might explain part of the normal glycerol levels obtained among the patients judged to be hyperthyroid. From the results, it is also evident that hypothyroid subjects cannot be distinguished from euthyroid by determining the glycerol concentration, which agrees with earlier findings’. A previous study8 has shown that the mean fractional turnover rate of glycerol for hyperthyroid patients was only increased by about 15% compared with normothyroid subjets. This points to a very slight influence of the hyperthyroid state on the fraction of glycerol eliminated. The mean blood glycerol concentration, however, was increased by 2100/~ and the mean glycerol turnover rate by 230%, which indicate a great enhancement of the fat mobilization in hyperthyroidism8. The hyperthyroid patients studied here in respect of the glycerol turnover were included in the previous study. When they were euthyroid on the carbimazole treatment, all the patients, except the obese woman discussed below, achieved a normal glycerol concentration and turnover rate. Also the mean fractional turnover rate decreased and became equal to that of the normothyroid group. The results indicate that, besides the disappearance of the signs and symptoms of hyperthyroidism, the fat mobilization is normalized in the euthyroid state during treatment. One patient in the carbimazole therapy group, a woman with moderate obesity, Clim.

C/vim

Acta,

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18g;Ig8

I97

PLASMA GLYCEROL IN HYPERTHYROIDISM

did not achieve a normal glycerol concentration on the treatment, although she became clinically and laboratorily euthyroid (Fig. I, case 6). Her glycerol turnover rate decreased, but was still above the normal level when euthyroid, indicating increased fat mobilization. Since she differed in this respect from the other patients, it is suggested that her enhanced lipolysis in the euthyroid state is perhaps due to her overweight. Although no correlation was made between the extent of hyperthyroid symptoms and glycerol concentration, it was generally found that patients with slight clinical symptoms had only a small elevation of the glycerol concentration. This may be exemplified by the four patients in Fig. 3, Group III, who were judged to be clinically euthyroid but laboratorily h~erthyroid, and by the h~erthyroid man shown in Fig. I (case 5). Their glycerol. concentrations ranged between upper normal value to slightly elevated. It is therefore concluded that only a moderate increase of the glycerol level can be expected in patients with slight clinical symptoms of hyperthyroidism, and a more marked elevation in patients with strong clinical evidence. CONCLUSIOK

The present results show that the determination of the glycerol concentration in blood provides information similar to that of conventional laboratory tests when examining for hyperthyroid function before and during antithyroid therapy. The advantage of the glycerol dete~ination is the simple and rapid technique for performing the analysis, and that capillary blood can be usedlo. Furthermore, the test can be used in subjects contaminated with iodine which gives misleading interpretations of the results of PBI and 1311uptake by the thyroid gland. The disadvantage is that, to obtain reliable results, the subject must submit to strictly standardized conditions in respect to fasting and rest when the blood collection is madea. Moreover, the blood samples have to be treated in order to avoid in vitro lipolysis of the triglyceride@. Other conditions involving altered lipid mobilization due to, e.g., prolonged fasting, excessive overweight, diabetes, or patients under treatment with drugs that interfere with sympathetic nervous activity, have to be excluded. Hypothyroid conditions cannot be revealed by this test.

This work was supported by grants from the Medical Faculty, LJniversity of Lund. Excellent technical assistance was given by Mrs. Gunvor Ekdahl and Mrs. Lisbet Nilsson. REFER.ENCES C. RICH, E. I..,. BIERMAN AND J. L. SCIIWARTZ, J.Chn. Invest., 38 (1959) 275. B. ET. MARKS, I. KIEM AND A. G. HTLLS, Metab.C&n. Ex$Z., g (1960) 1133. Clin.EnrZocpinol.Metab., 3 W. R.HARLAN, J. LASZT_~,M. D. BOGDONOFFANDE.K.ESTES,J~.,J. 23 (1963) 33. 4 F. SANDHOFER, S. SAILER AND H. BRAUNSTGINER, K&n. Wochsciav., 44 (1966) 433. 5 R. P.EATON, D. STEINBERGAND R. H. THOMPSON, J.CEin.Imesf.,~~ (1965) 247. b F. SANDHOFEK, S. SAILER AND H. BRAUNSTEINER, .Klira. Wachsch~~., 41 (1966) 1389. I

2

Clin. Chins. Acta,

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(1969) rag-rg8

TIBBLING

198 7 S. LAURELL AND G. TIBBLING, Clin. Chim. Acta, 24 (1968) 121. 8 G. TIBBLING, Clin. Chim. Acta, 24 (1969) IZI. g R. J. HAVEL, L. A. CARLSON, L.-G. EXELUND AND A. HOLMGREN, J. Appl.

Physiol.,

et d.

19 (1964)

613. IO II 12 13

S. LAURELL AND G. TIBBLING, Cl&. Chim. Acta, 13 (1966) 317. B. NOSSLIN, Stand. J. clin. Lab. Invest., 17, Suppl. 86 (1965) 177. International Atomic Energy Agency, Phys. ifi Med. Biol., 6 (1962) 533. L. A. CARLSON AND P. R. BALLY, in A. RENOLD AND G. CAHILL (Eds.), Handbook Adipose tissue, Williams and Wilkins, Baltimore, 1965, p. 557.

Clin. Chim.

Acta,

25 (1969) 189-198

ofPhysiology: