SERUM-THYROXINE AND THYROXINE-BINDING GLOBULIN IN SERIOUSLY ILL PATIENTS

208

antibodies against smooth-muscle were detectable in the sera at the beginning of the disease in titres of 40 to 640. The titres decreased gradually, and the test became negative 20-80 days after onset of the first symptoms. In 3 patients (nos. 3, 5, and 9) antinuclear antibodies became transiently detectable, but titres remained low. Of special interest were the results of measuring&bgr;1A. &bgr;1A levels first decreased, then rose above normal, and finally became normal. The lowest values of &bgr;1A coincided with the highest values of transaminase or followed these by a few days. In patient no. 5 &bgr;1A decreased in two distinct phases, the second coinciding with a renewed rise in transaminase. Discussion

So far as we know, s.H. antigen has not previously been found in faeces.’’ Cossart and Vahrman S reported on an antigen present in the faeces of patients with acute hepatitis, but this antigen was not related to s.H. antigen and s.H. antigen was not found in the sera of the patients.

points deserve further comment: patients studied, contact with injection (1) needles could be excluded. Nevertheless they had acquired an s.H.-antigen-positive hepatitis. In these cases fxces might have been the source of transmission. Krugman and Gilesfound that fasces of serologically s.H.-antigenpositive children could be used to transmit acute hepatitis. (2) Transfused blood containing S.H. antigen is more likely to transmit hepatitis than blood without s.H. antigen. 10 Our findings raise the question of the relationship between faecal s.H. antigen and transmission of hepatitis. Epidemiological studies are now under way to obtain further information on this point. Should evidence accumulate that individuals having faecal s.H. are a source of hepatitis, search for this antigen should become routine and special Some

SERUM-THYROXINE AND THYROXINEBINDING GLOBULIN IN SERIOUSLY ILL PATIENTS R. F. HARVEY Institute of Nuclear Medicine, Middlesex Hospital Medical School, London W1N 7RL

Serum-thyroxine, free thyroxine, and thyroxine-binding globulin (T.B.G.) were measured in thirty patients with severe medical illnesses. Many of the ill patients, especially those with fever, had raised free-thyroxine levels, but free thyroxine was normal or low in those with hypothermia. Mean T.B.G. capacity in the ill patients was significantly reduced when compared with that in healthy controls, the level being very low in a few In four patients the total thyroxine level was cases. abnormally low, and in all four T.B.G. capacity was notably reduced. It is suggested that the changes found may reflect, in part, a normal response to acute medical stress, as in the patients with fever, and in part a failing response, as in the patients with hypoSummary

thermia.

In 5 of the 11

rules of management should be established for the future. (3) Anti-s.H. antibodies were found in the faeces of 3 patients who formerly had had the corresponding S.H. antigen in their sera and faeces. Such antibodies were never found when antigen was still present in these media. This finding might have many implications: fxcal antibodies could account for immunity against fxcal infection, thus explaining why antibodies occur only rarely in serum. No data are so far available on this point. (4) Hepatitis associated with s.H. antigen is further characterised by the presence of antibodies against smoothmuscle,6 rarely of antinuclear antibodies, but also by a characteristic behaviour of &bgr;1A. The severest immunological abnormality seems to coincide with the peak of disease, while the appearance of s.H. antigen usually precedes the disease. These findings further support the hypothesis that not the virus itself but the immunological reaction of the host might be responsible for the symptoms. We thank Dr. A. M. Prince and Dr. B. S. Blumberg for providing reference sera; and Dr. M. Schmid, director of the Waid Hospital in Ziirich, and Dr. R. Braun, of the Clavadel Hospital in Davos, for kindly providing some of the test samples. Requests for reprints should be addressed to P. J. G. REFERENCES

Prince, A. M., Hargrove, R. L., Szmuness, W., Cherubini, C. E., Fontana, V. J., Jeffries, G. H. New Engl. J. Med. 1970, 282, 987. 2. Muller, J., Grob, P. Praxis (in the press). 3. Prince, A. M. Proc. natn. Acad. Sci. U.S.A. 1968, 60, 814. 4. Pesendorfer, F., Krassnitzky, O., Wewalka, F. Klin. Wschr. 1970, 48, 58. 1.

Introduction

SEVERAL endocrine and metabolic changes develop acute medical stress.1 Alterations in thyroid function are difficult to assess in such circumstances, because thyroid-hormone levels in serum depend not only on the function of the thyroid gland, but also on the level of thyroxine-binding globulin (T.B.G.), which may be altered in ill patients. Most of the thyroxine in serum is strongly bound to T.B.G. This protein-bound thyroxine is in equilibrium with a very small concentration of non-protein-bound (free) thyroxine, and it is this small fraction of the total serum-thyroxine which is thought to relate most closely to thyroid function.2 This fraction can be measured by dialysis (" free-thyroxine fraction ") and the free-thyroxine concentration can be obtained by multiplying the free fraction by the total serumthyroxine. Previous workers have shown that patients with chronic illnesses may have a decreased proteinbound iodine (P.B.I.) level 3,4 and an increased free thyroxine 5,6associated with changes in thyroxinebinding proteins.7 I report here a more detailed investigation of changes in patients with various types of severe medical illness.

during

Patients and Methods

Thirty seriously ill patients were investigated, sixteen and fourteen women (table I). Whenever possible

men

serum

was

obtained before

definitive

treatment

had

started, and no patient was taking drugs known to interfere with the binding of thyroxine by T.B.G. or to alter T.B.G. levels. The patients were arbitrarily divided into three 5. 6.

7. 8. 9. 10.

Mancini, G., Carbonara, A. O., Heremans, J. F. Immunochemistry, 1965, 2, 235. Farrow, L. J., Holborow, E. J., Johnson, G. D., Lamb, S. G., Stewart, J. S., Taylor, P. E., Zuckerman, A. J. Br. med. J. 1970, ii, 693. Ferris, A. A., Kaldor, J., Gust, I. D., Cross, G. Lancet, 1970, ii, 243. Cossart, Y. E., Vahrman, J. Br. med. J. 1970, i, 40. Krugman, S., Giles, J. P. J. Am. med. Ass. 1970, 212, 1019. Gocke, D. J., Kavey, N. B. Lancet, 1969, i, 1055.

209 TABLE

I—SERUM-THYROXINE, T.B.G. CAPACITY,

AND FREE-THYROXINE

LEVEL, AND CLINICAL DETAILS FOR 30 PATIENTS

WITH SEVERE MEDICAL ILLNESSES

groups according to body-temperature at the time the blood-sample was obtained. There were six patients with " hypothermia " (rectal temperature below 95 °F, 35 °C) and ten with " fever " (temperature above 101’F, 382°C),

the remainder having temperatures between 95 °F and 101 °F (" normothermic " group). Serum was also obtained from sixty healthy volunteers, thirty men and thirty women, none of whom had any evidence of thyroid or other diseases. Total serum-thyroxine was measured by the method of Ekins et al.,8 free-thyroxine fraction by the dialysis and magnesium-precipitation technique of Sterling and Brenner,9 and T.B.G. (as maximum thyroxine-binding capacity) by a modification 10 of the reverse-flow paperelectrophoresis method of Robbins.ll Results

Total

Thyroxine Fig. 1 shows the total serum-thyroxine level in the ill patients compared with healthy controls. Healthy women had a slightly but significantly higher thyroxine level than did the male controls (paired t test, t=325, Although there was no significant p<0.01). difference in mean thyroxine level, there was a significantly wider scatter of values among the ill patients. The total serum-thyroxine lay between 50 and 85 ng. per ml. in forty-nine of the sixty controls (82%) but in only twelve out of the thirty patients (40) The ill patients broadly fell (X2=5.78; P<0.02). into two groups, those with a tendency towards a raised serum-thyroxine and those whose thyroxine level was low. Thus the level was over 85 ng. per ml. in nine of thirty patients compared with eight of sixty controls, and under 50 ng. per ml. in nine patients compared with three controls (X2=12.0; P<0.001). Six of the nine patients with a thyroxine level over

Fig-1—Total serum-thyroxine levels in the ill patients compared with those in controls.

The values three groups.

are

shown in

ascending

order for each of the

85 ng. per ml. had fever, due to a variety of causes, but only one of the nine with a level below 50 ng. per ml. had fever: conversely three of these nine had hypothermia. Mean serum-thyroxine in ill patients with a "normal" temperature was 63-9 ng. per ml., in patients with hypothermia 49-5 ng. per ml., and in

210 TABLE II—MEAN (=S.D.) LEVELS OF THYROXINE, T.B.G. CAPACITY, AND FREE THYROXINE IN THE PATIENTS AND CONTROLS

those with fever 79-7 ng. per ml. (table II), the difference between those with hypothermia and those with fever being significant (P<0.05). Free Thyroxine Table 11 shows the mean levels of free thyroxine found in the controls and patients. There was no significant difference in free-thyroxine levels between male and female controls. The level was raised in many patients, especially those with fever, the mean in this group being significantly higher (P < 0.05) than that in the normothermic group. None of the patients had an abnormally low free-thyroxine concentration, even those with a low total thyroxine level, for in these the free-thyroxine fraction was invariably raised. All measurements of free-thyroxine fraction were made at 37 °C with no correction for the patient’s Had such a correction been made, temperature. several of the hypothermic patients would have had abnormally low levels (see Discussion).

T.B.G. The levels of T.B.G.

capacity found

in the ill

patients

shown in fig. 2. T.B.G. capacity was significantly higher in female than in male controls (s.E. of difference of two means 6-46, actual difference Mean T.B.G. capacity was lower 18-2, p<0.05). among the ill patients than among controls of either sex (comparing ill patients with normal men, s.E. of difference of two means 7-80, actual difference 17-1, This difference was mainly due to a few p<0.05). patients with extremely low T.B.G. capacity. These patients were usually gravely ill and had abnormally reduced total thyroxine levels (table r). Thus of the four patients in this series with total serum-thyroxine below 42-6 ng. per ml. (2 s.D. below normal mean) all had T.B.G. capacities more than 2 s.D. below the normal mean, and three died during the acute illness. There were no significant differences in T.B.G. levels between febrile normothermic and hypothermic patients. and controls

are

Relation between Total Thyroxine, T.B.G. Capacity, and Free-thyroxine Fraction Both in controls and in the ill patients the total thyroxine level was significantly related to T.B.G. in each case)-in women r=0-57; capacity (P<0-01 in men r==0-68; and in the patients r=0-58. Freethyroxine fraction was inversely related to T.B.G. women capacity in all the groups (P<0’01)—in r=-061; in men r=-0-82; and in patients r=-063.

Serum Albumin and Globulin To see whether alterations in T.B.G. capacity could be predicted from the serum-albumin or globulin levels, these were measured and compared with the corresponding T.B.G. capacity. No significant correlation was found between serum-albumin and T.B.G.

However, there was a capacity (r ==0-026, P>0.2). significant correlation between T.B.G. capacity and total serum-globulin (r==0-40, p<0.05), although the individual

scatter

of values

was

very wide.

Discussion

Fig. 2-T.B.G. capacity in the ill patients compared with that in controls.

The values groups.

are

shown in

ascending order

for each of the three

Although several workers 5-7,9, 12- 14 have demonstrated alterations in thyroxine-binding with a raised free-thyroxine fraction in patients with non-thyroid illnesses, the changes have not been studied in detail with regard to the type of illness. In some early investigations 6,13 the raised free-thyroxine fraction was thought to be due to a fall in the level of thyroxinebinding prealbumin, since no significant fall in T.B.G. capacity could be detected. However, these workers used standard electrophoresis to measure the capacities of the binding proteins, a method known to give less satisfactory results than reverse-flow electrophoresis when used for estimating T.B.G. capacity.ll Later workers, using reverse-flow electrophoresis, found a definite fall in T.B.G. capacity in ill patients.-, In these studies T.B.G. seemed to be the main determinant of free-thyroxine fraction, and this is what would be expected, since most of the serum-thyroxine (about 75%) is normally bound to T.B.G. with only a relatively small fraction bound to T.B.P.A. 15 My findings accord with this: free-thyroxine fraction was inversely related to T.B.G. capacity in controls and in the ill patients, as reported by Bellabarba et aI.7 Because of this relationship, when a low T.B.G.

211 was associated with a low serum-thyroxine, free-thyroxine fraction was raised and absolute free-thyroxine concentration normal (patients 1-3) or In most cases (patients 5-17, raised (patient 4). and total 19-22, 26) serum-thyroxine, T.B.G. capacity, and free-thyroxine concentration were all within the normal range. In the remaining patients (18, 23-25, and 27-30) free-thyroxine concentration was abnormally high. No difference in the types of underlying illness was noted between patients with these different findings, except that, as noted before, all four of the patients with abnormally low total thyroxine levels were gravely ill, and three of them died in the acute illness. However, when the patients were separated into those with fever, hypothermia, or a near-normal body-temperature, significant differences were seen. Mean serum-thyroxine was considerably higher in febrile than in hypothermic patients, and freethyroxine concentration was also greater (see table II). These findings are difficult to explain. Temperature plays a part in thyroxine binding,12 free-thyroxine fraction increasing with a rise in temperature and

capacity the

decreasing with a fall in temperature. Measurement of free-thyroxine fraction in this study was always performed at 37 °C, but this would have tended to lessen rather than exaggerate the changes found. Thus, in patients with fever, if free thyroxine had been measured at the same temperature as that of the patient when the blood-sample was taken, even higher free-thyroxine levels would have been found. In hypothermic patients, the free-thyroxine levels would have been lower than those observed, and this might well have brought the values into the " hypothyroid " range. Bernstein and Oppenheimer 12 found that a rise in dialysis temperature from 37 °C to 40 °C (104°F) increased measured free-thyroxine values by about 17%. Conversely, a fall in temperature from 370C to 350C (95 °F) decreased the measured freethyroxine values by over 25%. The raised freethyroxine concentration in febrile patients and the normal or low level in hypothermia could be either a primary or a secondary effect. Thus, cooling of the thyroid gland might be expected to reduce the output of thyroid hormones directly, which could accentuate any fall in serum-thyroxine. Since the normal halflife of thyroxine in serum is about 8 days, the hypothermic episode would probably have to be of at least a few days’ duration for this mechanism to produce significant changes, unless tissue utilisation of thyroxine was increased. Thyroxine and triiodothyronine are heat-producing hormones, by virtue of their effect on metabolism, and probably a more attractive hypothesis is that free-thyroxine levels are higher in febrile patients as one of the fever-producing mechanisms. Some failure of this response could result in a falling free-thyroxine level, with resulting hypothermia. Further studies in patients with hypothermia are clearly needed. Other

physiological factors may alter thyroxine in binding ill patients. Studying patients with hypothermia, Rosin and Exton-Smith 16 noted a raised T3 resin uptake (corresponding to an increased freethyroxine fraction) in many of their patients, and suggested that metabolic changes, such as acidosis, might be partly responsible. These factors were

further discussed by Rosin and Farran. 17 Any state in which serum-proteins are altered could influence thyroxine and T.B.G. levels. Thus in nephrotic syndrome 18 T.B.G. capacity and serum-thyroxine are reduced in proportion, with no resultant change in free-thyroxine level. The reverse changes might be expected in an underhydrated patient with a parallel rise in T.B.G. and serum-thyroxine. The manner of venepuncture may produce a similar effect if the bloodsample is taken after a period of stasis. Lewitus and Steinitz 19 found that a 5-minute period of venous occlusion before venepuncture raised P.B.I. levels by an average of 22% over those without occlusion. My results showed no correlation between T.B.G. capacity and serum-albumin, but there was a significant correlation of T.B.G. with serum-globulin levels. This relationship, however, was not sufficiently close for the serum-globulin to give a useful prediction of the T.B.G. capacity in an individual case. The poor correlation of T.B.G. capacity with serum-albumin level suggests that changes in plasma volume are not responsible for the alterations in T.B.G. capacity found in acutely ill patients, for if this were so parallel changes would be expected. A number of endogenous substances may affect serum-thyroxine and T.B.G. Free fatty acids 2and hormones such as adrenaline ’21 cortisone,22 and growth hormone 23 have all been shown to do so, and plasmalevels of all these substances may be altered in acute medical stress.1 In one series of twenty-five patients with accidental hypothermia, seven had a P.B.I. in the " hypothyroid " range and the initial plasma-11hydroxycorticosteroid level was higher in this group than in others in the series. 24This finding could be taken to indicate that those patients under the greatest degree of medical stress are the most likely to develop a low P.B.I, or serum-thyroxine, which accords with my results. Many patients with accidental hypothermia have severe underlying illnesses, and the clinical findings in accidental hypothermia may simulate myxœdema .25 It is likely that early studies of accidental hypothermia may well have overestimated the incidence of myxoedema as a cause, especially when the P.B.I. was used as the major criterion in the diagnosis and the patient died before any confirmatory tests could be performed. Some of these difficulties were discussed by Mathews .26 Acutely ill patients are often taking drugs, some of which may alter the binding of thyroxine by serumproteins. Drugs of this type include salicylates,27,28 32 2

None penicillin,29 prednisone,30,31 and heparin. of the patients in this study were having any of these drugs at the time the blood-samples were obtained, most being seen on admission or soon after. I suggest that in seriously ill patients, levels of thyroxine, free thyroxine, and T.B.G. capacity may all be altered, either as part of a stress response or because the underlying illness alters synthesis or metabolism of thyroxine and T.B.G. Total thyroxine or P.B.I. may fall within the hypothyroid " range, and free thyroxine, whether assessed by dialysis or indirect by a method such as the resin uptake of triiodothyronine,33 may be in the thyrotoxic range. It is often impracticable to carry out unrelated confirmatory "

"

"

212

of the thyroidal uptake of in ill radioiodine, patients. Attempts to diagacutely nose associated thyrotoxicosis or myxoedema in these patients should therefore be made with caution, preferably waiting until the acute illness has resolved.

tests, such

as measurement

9. 10. 11. 12. 13. 14.

I thank Dr. E. S. Williams, Dr. J. D. N. Nabarro, and Dr. R. P. Ekins for much helpful guidance; the physicians of the Middlesex Hospital, especially Dr. P. A. J. Ball, for allowing me to investigate their patients; and Dr. D. S. Sharland and Dr. A. N. Exton-Smith for allowing me to study patients with hypothermia admitted to the Whittington and St. Pancras Hospitals under their care. Some of the thyroxine assays were done by Mrs. S. Ellis, of the Institute of Nuclear Medicine, and measurements of serum albumin and globulin were made by staff of the Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, and their help is gratefully acknowledged. REFERENCES 1. 2.

3. 4. 5. 6.

7. 8.

Nabarro, J. D. N. Proc. R. Soc. Med. 1969, 62, 351. Robbins, J., Rail, J. E. in Hormones in Blood (edited by C. H. Gray and A. L. Bacharach); p. 383. New York, 1967. Kydd, D. M., Man, E. B., Peters, J. P. J. clin. Invest. 1950, 29, 1033. Engstrom, W. W., Markardt, B. J. clin. Endocr. Metab. 1955, 15, 953. Richards, J. B., Dowling, J. T., Ingbar, S. H. J. clin. Invest. 1959, 38, 1035. Oppenheimer, J. H., Squef, R., Surks, M. I., Hauer, H. ibid. 1963, 42, 1769. Bellabarba, D., Inada, M., Varsano-Aharon, N., Sterling, K. J. clin. Endocr. Metab. 1968, 28, 1023. Ekins, R. P., Williams, E. S., Ellis, S. Clin. Biochem. 1969, 2, 253.

Preliminary Communication INDIRECT IMMUNOFLUORESCENT STAINING OF ENTAMŒBA HISTOLYTICA IN TISSUES S. N. PARELKAR W. P. STAMM K. R. HILL Amœbiasis Research Unit, North Western Branch, Free Hospital, London N.W.3

Entamœba

Royal

in tissues can be indirect immunofluorescent technique which provides a rapid method of screening sections. If the method proves to be as specific as it seems to be, rapid diagnosis could be available even in specimens where no morphologically identifiable amœbæ can be seen.

Summary stained by

histolytica an

INTRODUCTION

THE identification of Entamceba histolytica in tissue is often difficult. The amoebae tend to be very unevenly distributed, so that several blocks of tissue may have to be sampled before the organisms are found. Furthermore, amoebae are difficult to identify positively since they can closely resemble macrophages or reticulum cells, and, unless the section has cut through the nucleus, identification is impossible. Goldman 1,2was the first to show that E. histolytica can be specifically stained by an immunofluorescence technique in preparations from cultures. Goldman et al. used the fluorescent-antibody technique to study the antigenic relationships of different strains of E. histolytica, and further applications of immunofluorescence to the antigenic analysis of E. histolytica strains have been reported by Goldman and his

15. 16. 17. 18.

19. 20. 21. 22. 23. 24.

Sterling, K., Brenner, M. A. J. clin. Invest. 1966, 45, 153. Tanaka, S., Starr, P. J. clin. Endocr. Metab. 1959, 19, 84. Robbins, J. Archs Biochem. 1956, 63, 461. Bernstein, G., Oppenheimer, J. H. J. clin. Endocr. Metab. 1966, 26, 195. Ingbar, S. H., Braverman, L. E., Dawber, N. A., Lee, G. Y. J. clin. Invest. 1965, 44, 1679. Arango, G., Mayberry, W. E., Hockert, T. J., Elveback, L. R. Proc. Staff Meet. Mayo Clin. 1968, 43, 503. Woeber, K. A., Ingbar, S. H., Traenkle, U. I. J. clin. Invest. 1968, 47, 1710. Rosin, A. J., Exton-Smith, A. N. Br. med. J. 1964, i, 16. Rosin, A. J., Farran, H. E. J. Am. Geriat. Soc. 1968, 16, 1030. Robbins, J., Rail, J. E., Petermann, M. L. J. clin. Invest. 1957, 36, 1333. Lewitus, Z., Steinitz, K. Clinica chim. Acta, 1963, 8, 629. Braverman, L. E., Arky, R. A., Foster, A. E., Ingbar, S. H. J. clin. Invest. 1969, 48, 878. Hays, M. T., Solomon, D. H. ibid. 1969, 48, 1114. Blomstedt, B., Einhorn, J. Metabolism, 1967, 16, 319. Oliner, L., Ballantine, J. J. J. clin. Endocr. Metab. 1968, 28, 603. Sprunt, J. G., Maclean, D., Browning, M. C. K. Lancet, 1970, i,

324. 25. Hockaday, T. D. R. Br. J. Hosp. Med. 1969, 2, 1083. 26. Mathews, J. A. Postgrad. med. J. 1966, 42, 495. 27. Woeber, K. A., Ingbar, S. H. Endocrinology, 1963, 73, 118. 28. Musa, B. U., Kumar, R. S., Dowling, J. T. J. clin. Endocr. Metab. 1968, 28, 1461. 29. Surks, M. I., Oppenheimer, J. H. Endocrinology, 1963, 72, 567. 30. Oppenheimer, J. H., Werner, S. C. J. clin. Endocr. Metab. 1966, 26, 715. 31. Kumar, R. S., Musa, B. U., Appleton, W. G., Dowling, J. T. ibid. 1968, 28, 1335. 32. Schatz, D. L., Sheppard, R. H., Steiner, G., Chandarlapaty, C. S., DeVeber, G. A. ibid. 1969, 29, 1015. 33. Clark, F., Horn, D. B. ibid. 1965, 25, 39.

colleagues.4.sThese workers used the direct technique for fluorescent antibody staining; the indirect or " sandwich " technique has been applied to the immunofluorescence detection of amoebic serum antibodies by several workers. 6-9 There

reports so far of immunofluorescent used for the detection of E. histolytica staining being in tissues, although Doxiades and Candreviotis 10 proposed that this should be done. Conventional paraffin sections prepared from tissues fixed in formalin are convenient but have not been extensively used for fluorescent-antibody studies because many antiSome antigens gens are destroyed in the process. (e.g., certain mucins or microorganisms) may be detected by immunofluorescence in old routine paraffin blocks of formalin-fixed surgical biopsy or necropsy material. 11 are no

Most work on histological material has been done by direct staining with a conjugated antiserum. The indirect sandwich technique has the great advantage that the same conjugated immunoglobulin can be used with any antibody and is easily available commercially. This technique is also substantially more sensitive than single-layer tracing.ll For these reasons we decided to attempt specific immunofluorescent staining of E. histolytica in tissues by the indirect technique. MATERIALS AND METHODS

Specific Immune Sera The immune sera were kindly supplied by Dr. A. L. Jeanes, of Guy’s Hospital; they were sera which he had found to have a high titre by the amoebic immunofluorescent-antibody test. Five sera were titred using tissue sections known to contain amoebæ. The titre adopted for routine staining was one dilution lower than the highest dilution which still gave maximum staining.