- Email: [email protected]
HETEROGENEITY OF HUMAN GROWTH HORMONE ITS INFLUENCE ON A RADIO-IMMUNOASSAY OF THE HORMONE IN SERUM
1403
Using the same principle for measuring cerebral 30 32 and myocardial 35 blood-flow, the results have been reliable. Concerning the renal blood-flow, Thorburn et al.34 found the expected normal values in the dog. Our normotensive patient (case 4) likewise exhibited near " normal " values (about 4 ml. per g. per minute). It seems possible to determine whether the human Goldblatt kidney of necessity shows " ischsemia ". The method also seems suitable for determining renal bloodflow when the clearance methods are known to be misleading-for instance in cases of" shock "kidney ". Although technically resembling 1-hippuran renography " for divided kidney function assessment it must be emphasised that the two methods are fundamentally different. In this preliminary communication we have not considered the possibility of separately determining the different components of the medullary blood-flow by dissolving the curves. The work of Thorburn et al.34 makes this separation possible. SUMMARY
A new method for determination of divided renal blood-flow in man is described. The principle of the method consist of the injection of a radioactive inert gas (e.g., krypton-85) into the renal artery and external monitoring of the disappearance of the isotope from the
kidney. We wish
to
thank Dr. N. A. Lassen for advice and encouragement.
E. KEMP
Copenhagen K. HØEDT-RASMUSSEN M.D. Copenhagen J. K. BJERRUM M.D. Copenhagen A. FAHRENKRUG M.D. Copenhagen M.D.
Medical Department B, Bispebjerg Hospital, and X-ray Department,
Bispebjerg Bakke 23, Copenhagen N.V., Denmark
J. LADEFOGED M.D. Aarhus
HETEROGENEITY OF HUMAN GROWTH HORMONE ITS INFLUENCE ON A RADIO-IMMUNOASSAY OF THE HORMONE IN SERUM
METHODS for the estimation of human growth hormone (H.G.H.) in serum, such as the hsemagglutination-inhibition technique of Read and Bryan 36 and the radio-immunoassays described by Greenspan et a1.3’ and Utiger et al.38 are influenced by non-specific factors present in serum, though Hunter and Greenwood 39 when describing their radio-immunoassay for H.G.H. did not mention any such influence. We have found evidence that heterogeneity of currently available H.G.H. preparations may contribute to the difficulties encountered in immunoassays, and that the specificity of the method can be enhanced by fractionation of the H.G.H.-13’I by means of gel filtration. METHODS
(Raben preparation, 1.5 U.S.P. units per mg.) was radioiodinated according to the method of Hunter and Greenwood . 40 The labelled hormone (specific activity 200-400 mC per mg.) was found to be immunologically identical with unlabelled H.G.H. H.G.H.
35. 36. 37.
38. 39. 40.
Herd, J. A., Hollenberg, M., Thornburn, G. D., Kopald, H. H., Barger, A. C. Amer. J. Physiol. 1962, 203, 122. Read, C. H., Bryan, G. T. Recent Progr. Hormone Res. 1960, 16, 187. Greenspan, F. S., Cofell, J. A., Lew, W., Peng, C. T. J. Lab. clin. Med. 1962, 59, 520. Utiger, R. D., Parker, M. L., Daughaday, W. H. J. clin. Invest. 1962, 41, 254. Hunter, W. M., Greenwood, F. C. Acta endocr., Copenhagen, 1962, suppl. 67, p. 59. Hunter, W. M., Greenwood, F. C. Nature, Lond. 1962, 194, 495.
Rabbit hormone
H.G.H. as
antiserum, reacting specificially
demonstrated in
with the
immunoelectrophoresis and
hasmagglutination-inhibition tests, was shown to inhibit its biological activity in the hypophysectomised rat (tibia test). found also to contain antibodies to human were removed by absorption. No antibodies could be demonstrated to other serum components. Antiserum dilutions and standard solutions of H.G.H. were prepared in 1% normal rabbit serum in 0.01 M phosphate/1 0-15 M NaCl buffer at pH 7-2 with merthiolate (final concentration 1/10,000) and kept at 4°C. No losses of immunological potency of both standard H.G.H. solutions and antiserum dilutions could be demonstrated after nine months of storage at 4°C. Tracer amounts of H.G.H._131I (1-2 mug.) were incubated with different dilutions of antiserum (10-4’°-10-5’°) for three days at 4°C, and the dilution giving approximately 70% binding of the labelled hormone to immune gamma-globulin was chosen for the assay (usually 10-4’3). Addition of increasing amounts of unlabelled H.G.H. to the assay system led to a progressive diminution of the proportion of H.G.H.-131I bound to immune The antiserum
was
serum-albumin; these
gamma-globulin (% bound). A standard curve was
obtained
by plotting the of added unlabelled H.G.H. against the percentage of bound H.G.H.-232I (fig. 1). Separation of the H.G.H..131 bound amount
to
gamma-globulin
from the free H.G.H.131 was achieved by agar-gel electrophoresis. The agar
(Special Agar Noble Difco) was washed in distilled water for two days to reduce electroendosmosis, thus Fig. 1-Standard curve of H.G.H. radioimmunoassay. achieving a clear separation of the x2-globulin fraction from the origin. This gave the best separation of the bound from the free H.G.H.-131I, because the latter moves as a slow
Gel filtration of H.G.H.-131I without serum gave an elution pattern as shown in fig. 2. Fractions 1 to 5 belong to the void volume of the column ; any proteins therein are excluded from the gel and therefore must have relatively large molecular size (molecular weight presumably > 200,000). H.G.H.131I is clearly not homogeneous as regards molecular size; this is 41. 42.
Smithies, O. Biochem. J. 1959, 71, 585. Ferguson, K. A., Wallace, A. L. C. Nature, Lond. 1961, 190, 629.
1404
addition of both human and animal serum to the incubation mixture altered the distribution of the radioactivity in agar-gel electrophoresis and in gel filtration. The technique is thus influenced by serum-proteins. We found evidence that the proteins responsible are (X2 and &bgr;2 macroglobulins rather than the lipoproteins. These macroglobulins have electrophoretic mobilities in agar between those of H.G.H._131I bound to gammaglobulin and free H.G.H._131I, and they thus prevent exact estimation of these two H.G.H._131I fractions. It has been shown and we have confirmed, that G.H. consists of at least three components demonstrable Fig. 2-Sephadex G 200 gel filtration of 50 m[tg. H.G.H.-13’I in 1 ml. electrophoresis on both starch gel and cellulose acetate; barbital buffer (flow-rate 2 ml. per hour). all three components react strongly with H.G.H. antiserum. was shown by probably not due to the labelling of the hormone because the The hormonal activity of these components
by
preparation (unlabelled) gives a comparable elution pattern when optical density (280 m is used as a measure of protein concentration. The discrepancies between the optical-density curve and the radioactivity curve are probably due to differences in the degree of protein iodination of the fractions, the optical-density curve showing a higher proportion of the high molecular-weight same
Ferguson and Wallace.45
fractions. The addition of serum to H.G.H.-131J before gel filtration increased the radioactivity of the first fractions from 4 to 11 % of the total radioactivity (fig. 3), the peak coinciding with the first main peak of the serum-proteins (fractions 1 to 5). Immunoelectrophoresis showed that this first peak contained the macromolecular substances present in serum-i.e., OC2-macroglobulin, &bgr;a-macroglobulin, the «-(3 lipoproteins, and an unidentified CX2-globulin. This is ’in agreement with the findings of Flodin and Killander. 43 The second serum-protein peak (fractions 9 to 11) was mainly composed of gamma-
globulin (molecular weight 150,000) and the last (fractions 20 to 22) contained mainly albumin (molecular weight 69,000). Elution of the radioactivity was maximal after the albumin peak; this agrees with the stated molecular weight of H.G.H. (27,000).44 The radioactivity elution pattern was not significantly altered by increasing the amount of H.G.H._131I per volume of serum,’indicating that saturation of a possible specific H.G.H.-carrier protein is unlikely. The results of testing the various fractions for immunological activity are shown in fig. 4. DISCUSSION
The radio-immunoassay for H.G.H. is based on the assumption that only immune gamma-globulin can bind H.G.H._131I. But even in the absence of H.G.H. antiserum, 43. 44.
Flodin, P., Killander, J. Biochem. biophys. Acta, 1962, 63, 403. Squire, P. G., Pedersen, K. O. J. Amer. chem. Soc. 1961, 83, 476.
Fig. 4-Immunological activity of gel-filtration fractions of H.G.H.-132I, as determined in agar-gel electrophoresis. Continuous lines represent fractions from gel filtration of H.G.H.-tt!.} alone, and broken lines represent fractions from gel filtration of H.G.H._1811 in
serum.
By gel filtration of H.G.H._131I with and without
serum
found that the three components are eluted in the fractions with a low molecular weight (fractions 20 to 29 of the experiments shown in figs. 2 and 3), and that they are immunologically very active (fig. 4). In addition, starch-gel electrophoresis of fractions 2 to 7 (figs. 2 and 3) demonstrated that most of the radioactivity is found in regions corresponding to the P2 and OC2 serummacroglobulins-i.e., the origin and about 1 cm. from the origin on the anodic side, respectively. In agar electrophoresis about half of these fractions are localised on the cathodic side of the gel, which explains the high proportion of non-specific binding of these fractions in the radio-immunoassay in the absence of antiserum (fig. 4). The addition of antiserum did not alter this proportion, indicating that these fractions have
we
immunological activity. our opinion, only the fractions of low molecular weight (which are not bound to serum-proteins) are hormonally and immunologically active, and should be called H.G.H., and the fractions with a high molecular
no
In
Fig. 3-Sephadex normal human
G 200
gel filtration of 50 mg. H.G.H.-1311 in 1 ml (flow-rate 2 ml. per hour).
serum
45. 46.
Ferguson, K. A., Wallace, A. L. C. Nature,Lond. 1961, 190, 632. Barrett, R. J., Friesen, H., Astwood, E. B. J. biol. Chem. 1962, 237, 432.
1405
weight should be regarded as impurities. The latter can influence immunoassay procedures, as they can be bound to serum-proteins. There is no reason to think that the H.G.H. preparation used differs significantly with regard to impurities from other H.G.H. preparations; Irie and Barrett,47 using starchgel electrophoresis and gel filtration, also found evidence of contaminants in their H.G.H. preparations. The availability of pure or nearly pure H.G.H. is a prerequisite for any reliable immunoassay of H.G.H. in serum. Accuracy can be improved, however, by using H.G.H._1311 which has been purified by gel filtration. SUMMARY
Evidence is presented which indicates that human growth hormone (H.G.H., Raben) contains impurities which interfere with immunoassays of H.G.H. in serum. These impurities can to a large extent be removed by gel filtration, thus enhancing the specificity of the method. We are indebted to Dr. J. D. H. Homan and Dr. E. de Jager of N.V. Organon, Oss, The Netherlands, for a supply of hormone preparations and for performing the tibia tests. It is a pleasure to acknowledge the assistance and helpful criticism of Prof. J. J. van Loghem and Dr. H. W. Krijnen. J. L. TOUBER Department of Radiochemistry, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam
M.D.
Amsterdam
D. MAINGAY M.D.
Leyden
ADDENDUM
Since this article was - submitted for publication Reisfeld et al. (R. A. Reisfeld, B. G. Hallows, D. E. Williams, N. G. Brink, S. L. Steelman. Nature, Lond. 1963, 197, 1206) have reported similar results of purifying H.G.H. on Sephadex G 200, obtaining a product with a biological potency of 2-2-5 U.S.P. units per mg. Our results and those of Reisfeld et al. indicate that the current practice of stating amounts of (Raben) H.G.H. in weight units should be replaced by stating the amounts in units of biological activity (i.e., U.S.P. units per mg. protein). EFFECT OF MAGNESIUM ON THE THYROID THYROID disease is accompanied by disturbances in
effect was absent when the magnesium reached 24 micromoles per ml. The antagonistic action of magnesium appears to be specific to thyroxine since it is absent when uncoupling is produced by other factors. 52 It could be deduced from the above that magnesium would antagonise the effect of thyroxine at subcellular (mitochondrial) level, and therefore it was decided to try the effect of giving parenteral magnesium to patients with hyperthyroidism. Two patients who had been admitted to hospital with hyperthyroidism and assessed for seven days were given 20 ml. of a 25% solution of magnesium sulphate by deep intramuscular injection. The injections were painful, and after one week the condition of each patient remained largely unchanged both clinically and by laboratory assessment including B.M.R. and protein-bound
thyroxine
iodine. An attempt was then made to use magnesium chloride instead of sulphate since it was suggested that sulphate ions might speed excretion of the magnesium. In the first 1 % solution of magnesium chloride was used, but present a 14% solution is used and injected in amounts of 20 ml. Three patients with hyperthyroidism have been treated with daily injections for from three to seven weeks, and all showed a diminution in the size of the thyroid and The third some improvement in clinical condition. in the diminution patient had a sudden and remarkable circumference of the neck (12 cm.) with an accompanying disappearance of his toxic symptoms. Two patients with non-toxic goitre were treated, one having been deaf since birth, and both of these patients showed a remarkable diminution in the size of the thyroid gland. Microscopic examination has not revealed any specific change in the
place
glands removed. Although it is not known how magnesium produces its effect, it seems worth while making a preliminary report since a high level of magnesium can apparently cause notable diminution in the size of the thyroid gland both in toxic and in non-toxic goitre. The first two patients were treated in London under the Mr. Selwyn Taylor and the other five in Cairo. Ains Shams
University Hospital, Cairo, Egypt, United Arab Republic
magnesium metabolism.
The normal magnesium content of the serum is 1-3-5 mg. per 100 ml., 20% being proteinbound and the remainder ionised. Dine and Lavietes 48 showed that in myxoedema all the magnesium was ionised while in hyperthyroidism up to half was protein-bound. Tapley 49 reported that administration of triiodothyronine to patients with myxoedema caused a rise in the urinary excretion of magnesium. It is likely that magnesium antagonises the effect of thyroxine on oxidation in the cells. Normally, energy released by oxidation in the cells is partly converted into heat and partly stored as energy-rich phosphate bonds in adenosine triphosphate. This coupling takes place in the mitochondria, and uncoupling results in a raised basal metabolic rate (B.M.R.). It can be produced by thyroxine and dinitrophenol. Feldott and Lardy 50 showed that, in vitro, thyroxine uncouples oxidative phosphorylation. Brody et al.51 showed that the uncoupling action of thyroxine was enhanced when the magnesium level fell below 12 micromoles per ml. and was r.eversed with magnesium levels above this. The 47. 48. 49. 50. 51.
Irie, M., Barrett, R. J. Endocrinology, 1962, 71, 277. Dine, R. F., Lavietes, P. H. J. clin. Invest. 1942, 21, 781. Tapley, D. F. J. biol. Chem. 1956, 222, 325. Feldott, G., Lardy, H. A. ibid. 1951, 192, 447. Brody, T. M., Hurwitz, R., Bain, J. A. Antibiot. and Chemother. 1954, 4, 864.
a
at
care
of
M. A. NEGUIB M.S.
Cairo
Medical Societies BRITISH ORTHOPÆDIC ASSOCIATION THE spring meeting of the British Orthopeadic Association was held in Sheffield on May 16-18. Mr. F. W. HOLDSWORTH, the President, was in the chair. We summarise below a few of the contributions. Mr. SIDNEY PAPPWORTH (Sheffield), in a clinical demonstration on subtrochanteric osteotomy as primary treatment of fractures of the femoral neck, said that nailing subcapital fractures of the femoral neck resulted in 25% of success only. For the past ten years he had combined reduction of the fracture with a McMurray type of osteotomy and fixation with a nail plate, the nail being introduced through the cut end of the bone. The osteotomy must be done as described by McMurray with the shaft displaced to support the femoral head. Then it did not matter whether the fracture united or the femoral head became avascular, and patients were still able to walk in comfort. Mr. J. R. KIRKUP (Bath) reported 84 patients treated similarly with a follow-up of six months to six years. All the osteotomies united; 39% had non-union, of the fracture; and 32% had avascular necrosis. Patients walked on the avascular heads without pain. The procedure was particularly indicated 52.
Wiswell, J. G., Braverman, M. G. Endocrinology, 1957, 61,
153.
Using the same principle for measuring cerebral 30 32 and myocardial 35 blood-flow, the results have been reliable. Concerning the renal blood-flow, Thorburn et al.34 found the expected normal values in the dog. Our normotensive patient (case 4) likewise exhibited near " normal " values (about 4 ml. per g. per minute). It seems possible to determine whether the human Goldblatt kidney of necessity shows " ischsemia ". The method also seems suitable for determining renal bloodflow when the clearance methods are known to be misleading-for instance in cases of" shock "kidney ". Although technically resembling 1-hippuran renography " for divided kidney function assessment it must be emphasised that the two methods are fundamentally different. In this preliminary communication we have not considered the possibility of separately determining the different components of the medullary blood-flow by dissolving the curves. The work of Thorburn et al.34 makes this separation possible. SUMMARY
A new method for determination of divided renal blood-flow in man is described. The principle of the method consist of the injection of a radioactive inert gas (e.g., krypton-85) into the renal artery and external monitoring of the disappearance of the isotope from the
kidney. We wish
to
thank Dr. N. A. Lassen for advice and encouragement.
E. KEMP
Copenhagen K. HØEDT-RASMUSSEN M.D. Copenhagen J. K. BJERRUM M.D. Copenhagen A. FAHRENKRUG M.D. Copenhagen M.D.
Medical Department B, Bispebjerg Hospital, and X-ray Department,
Bispebjerg Bakke 23, Copenhagen N.V., Denmark
J. LADEFOGED M.D. Aarhus
HETEROGENEITY OF HUMAN GROWTH HORMONE ITS INFLUENCE ON A RADIO-IMMUNOASSAY OF THE HORMONE IN SERUM
METHODS for the estimation of human growth hormone (H.G.H.) in serum, such as the hsemagglutination-inhibition technique of Read and Bryan 36 and the radio-immunoassays described by Greenspan et a1.3’ and Utiger et al.38 are influenced by non-specific factors present in serum, though Hunter and Greenwood 39 when describing their radio-immunoassay for H.G.H. did not mention any such influence. We have found evidence that heterogeneity of currently available H.G.H. preparations may contribute to the difficulties encountered in immunoassays, and that the specificity of the method can be enhanced by fractionation of the H.G.H.-13’I by means of gel filtration. METHODS
(Raben preparation, 1.5 U.S.P. units per mg.) was radioiodinated according to the method of Hunter and Greenwood . 40 The labelled hormone (specific activity 200-400 mC per mg.) was found to be immunologically identical with unlabelled H.G.H. H.G.H.
35. 36. 37.
38. 39. 40.
Herd, J. A., Hollenberg, M., Thornburn, G. D., Kopald, H. H., Barger, A. C. Amer. J. Physiol. 1962, 203, 122. Read, C. H., Bryan, G. T. Recent Progr. Hormone Res. 1960, 16, 187. Greenspan, F. S., Cofell, J. A., Lew, W., Peng, C. T. J. Lab. clin. Med. 1962, 59, 520. Utiger, R. D., Parker, M. L., Daughaday, W. H. J. clin. Invest. 1962, 41, 254. Hunter, W. M., Greenwood, F. C. Acta endocr., Copenhagen, 1962, suppl. 67, p. 59. Hunter, W. M., Greenwood, F. C. Nature, Lond. 1962, 194, 495.
Rabbit hormone
H.G.H. as
antiserum, reacting specificially
demonstrated in
with the
immunoelectrophoresis and
hasmagglutination-inhibition tests, was shown to inhibit its biological activity in the hypophysectomised rat (tibia test). found also to contain antibodies to human were removed by absorption. No antibodies could be demonstrated to other serum components. Antiserum dilutions and standard solutions of H.G.H. were prepared in 1% normal rabbit serum in 0.01 M phosphate/1 0-15 M NaCl buffer at pH 7-2 with merthiolate (final concentration 1/10,000) and kept at 4°C. No losses of immunological potency of both standard H.G.H. solutions and antiserum dilutions could be demonstrated after nine months of storage at 4°C. Tracer amounts of H.G.H._131I (1-2 mug.) were incubated with different dilutions of antiserum (10-4’°-10-5’°) for three days at 4°C, and the dilution giving approximately 70% binding of the labelled hormone to immune gamma-globulin was chosen for the assay (usually 10-4’3). Addition of increasing amounts of unlabelled H.G.H. to the assay system led to a progressive diminution of the proportion of H.G.H.-131I bound to immune The antiserum
was
serum-albumin; these
gamma-globulin (% bound). A standard curve was
obtained
by plotting the of added unlabelled H.G.H. against the percentage of bound H.G.H.-232I (fig. 1). Separation of the H.G.H..131 bound amount
to
gamma-globulin
from the free H.G.H.131 was achieved by agar-gel electrophoresis. The agar
(Special Agar Noble Difco) was washed in distilled water for two days to reduce electroendosmosis, thus Fig. 1-Standard curve of H.G.H. radioimmunoassay. achieving a clear separation of the x2-globulin fraction from the origin. This gave the best separation of the bound from the free H.G.H.-131I, because the latter moves as a slow
Gel filtration of H.G.H.-131I without serum gave an elution pattern as shown in fig. 2. Fractions 1 to 5 belong to the void volume of the column ; any proteins therein are excluded from the gel and therefore must have relatively large molecular size (molecular weight presumably > 200,000). H.G.H.131I is clearly not homogeneous as regards molecular size; this is 41. 42.
Smithies, O. Biochem. J. 1959, 71, 585. Ferguson, K. A., Wallace, A. L. C. Nature, Lond. 1961, 190, 629.
1404
addition of both human and animal serum to the incubation mixture altered the distribution of the radioactivity in agar-gel electrophoresis and in gel filtration. The technique is thus influenced by serum-proteins. We found evidence that the proteins responsible are (X2 and &bgr;2 macroglobulins rather than the lipoproteins. These macroglobulins have electrophoretic mobilities in agar between those of H.G.H._131I bound to gammaglobulin and free H.G.H._131I, and they thus prevent exact estimation of these two H.G.H._131I fractions. It has been shown and we have confirmed, that G.H. consists of at least three components demonstrable Fig. 2-Sephadex G 200 gel filtration of 50 m[tg. H.G.H.-13’I in 1 ml. electrophoresis on both starch gel and cellulose acetate; barbital buffer (flow-rate 2 ml. per hour). all three components react strongly with H.G.H. antiserum. was shown by probably not due to the labelling of the hormone because the The hormonal activity of these components
by
preparation (unlabelled) gives a comparable elution pattern when optical density (280 m is used as a measure of protein concentration. The discrepancies between the optical-density curve and the radioactivity curve are probably due to differences in the degree of protein iodination of the fractions, the optical-density curve showing a higher proportion of the high molecular-weight same
Ferguson and Wallace.45
fractions. The addition of serum to H.G.H.-131J before gel filtration increased the radioactivity of the first fractions from 4 to 11 % of the total radioactivity (fig. 3), the peak coinciding with the first main peak of the serum-proteins (fractions 1 to 5). Immunoelectrophoresis showed that this first peak contained the macromolecular substances present in serum-i.e., OC2-macroglobulin, &bgr;a-macroglobulin, the «-(3 lipoproteins, and an unidentified CX2-globulin. This is ’in agreement with the findings of Flodin and Killander. 43 The second serum-protein peak (fractions 9 to 11) was mainly composed of gamma-
globulin (molecular weight 150,000) and the last (fractions 20 to 22) contained mainly albumin (molecular weight 69,000). Elution of the radioactivity was maximal after the albumin peak; this agrees with the stated molecular weight of H.G.H. (27,000).44 The radioactivity elution pattern was not significantly altered by increasing the amount of H.G.H._131I per volume of serum,’indicating that saturation of a possible specific H.G.H.-carrier protein is unlikely. The results of testing the various fractions for immunological activity are shown in fig. 4. DISCUSSION
The radio-immunoassay for H.G.H. is based on the assumption that only immune gamma-globulin can bind H.G.H._131I. But even in the absence of H.G.H. antiserum, 43. 44.
Flodin, P., Killander, J. Biochem. biophys. Acta, 1962, 63, 403. Squire, P. G., Pedersen, K. O. J. Amer. chem. Soc. 1961, 83, 476.
Fig. 4-Immunological activity of gel-filtration fractions of H.G.H.-132I, as determined in agar-gel electrophoresis. Continuous lines represent fractions from gel filtration of H.G.H.-tt!.} alone, and broken lines represent fractions from gel filtration of H.G.H._1811 in
serum.
By gel filtration of H.G.H._131I with and without
serum
found that the three components are eluted in the fractions with a low molecular weight (fractions 20 to 29 of the experiments shown in figs. 2 and 3), and that they are immunologically very active (fig. 4). In addition, starch-gel electrophoresis of fractions 2 to 7 (figs. 2 and 3) demonstrated that most of the radioactivity is found in regions corresponding to the P2 and OC2 serummacroglobulins-i.e., the origin and about 1 cm. from the origin on the anodic side, respectively. In agar electrophoresis about half of these fractions are localised on the cathodic side of the gel, which explains the high proportion of non-specific binding of these fractions in the radio-immunoassay in the absence of antiserum (fig. 4). The addition of antiserum did not alter this proportion, indicating that these fractions have
we
immunological activity. our opinion, only the fractions of low molecular weight (which are not bound to serum-proteins) are hormonally and immunologically active, and should be called H.G.H., and the fractions with a high molecular
no
In
Fig. 3-Sephadex normal human
G 200
gel filtration of 50 mg. H.G.H.-1311 in 1 ml (flow-rate 2 ml. per hour).
serum
45. 46.
Ferguson, K. A., Wallace, A. L. C. Nature,Lond. 1961, 190, 632. Barrett, R. J., Friesen, H., Astwood, E. B. J. biol. Chem. 1962, 237, 432.
1405
weight should be regarded as impurities. The latter can influence immunoassay procedures, as they can be bound to serum-proteins. There is no reason to think that the H.G.H. preparation used differs significantly with regard to impurities from other H.G.H. preparations; Irie and Barrett,47 using starchgel electrophoresis and gel filtration, also found evidence of contaminants in their H.G.H. preparations. The availability of pure or nearly pure H.G.H. is a prerequisite for any reliable immunoassay of H.G.H. in serum. Accuracy can be improved, however, by using H.G.H._1311 which has been purified by gel filtration. SUMMARY
Evidence is presented which indicates that human growth hormone (H.G.H., Raben) contains impurities which interfere with immunoassays of H.G.H. in serum. These impurities can to a large extent be removed by gel filtration, thus enhancing the specificity of the method. We are indebted to Dr. J. D. H. Homan and Dr. E. de Jager of N.V. Organon, Oss, The Netherlands, for a supply of hormone preparations and for performing the tibia tests. It is a pleasure to acknowledge the assistance and helpful criticism of Prof. J. J. van Loghem and Dr. H. W. Krijnen. J. L. TOUBER Department of Radiochemistry, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam
M.D.
Amsterdam
D. MAINGAY M.D.
Leyden
ADDENDUM
Since this article was - submitted for publication Reisfeld et al. (R. A. Reisfeld, B. G. Hallows, D. E. Williams, N. G. Brink, S. L. Steelman. Nature, Lond. 1963, 197, 1206) have reported similar results of purifying H.G.H. on Sephadex G 200, obtaining a product with a biological potency of 2-2-5 U.S.P. units per mg. Our results and those of Reisfeld et al. indicate that the current practice of stating amounts of (Raben) H.G.H. in weight units should be replaced by stating the amounts in units of biological activity (i.e., U.S.P. units per mg. protein). EFFECT OF MAGNESIUM ON THE THYROID THYROID disease is accompanied by disturbances in
effect was absent when the magnesium reached 24 micromoles per ml. The antagonistic action of magnesium appears to be specific to thyroxine since it is absent when uncoupling is produced by other factors. 52 It could be deduced from the above that magnesium would antagonise the effect of thyroxine at subcellular (mitochondrial) level, and therefore it was decided to try the effect of giving parenteral magnesium to patients with hyperthyroidism. Two patients who had been admitted to hospital with hyperthyroidism and assessed for seven days were given 20 ml. of a 25% solution of magnesium sulphate by deep intramuscular injection. The injections were painful, and after one week the condition of each patient remained largely unchanged both clinically and by laboratory assessment including B.M.R. and protein-bound
thyroxine
iodine. An attempt was then made to use magnesium chloride instead of sulphate since it was suggested that sulphate ions might speed excretion of the magnesium. In the first 1 % solution of magnesium chloride was used, but present a 14% solution is used and injected in amounts of 20 ml. Three patients with hyperthyroidism have been treated with daily injections for from three to seven weeks, and all showed a diminution in the size of the thyroid and The third some improvement in clinical condition. in the diminution patient had a sudden and remarkable circumference of the neck (12 cm.) with an accompanying disappearance of his toxic symptoms. Two patients with non-toxic goitre were treated, one having been deaf since birth, and both of these patients showed a remarkable diminution in the size of the thyroid gland. Microscopic examination has not revealed any specific change in the
place
glands removed. Although it is not known how magnesium produces its effect, it seems worth while making a preliminary report since a high level of magnesium can apparently cause notable diminution in the size of the thyroid gland both in toxic and in non-toxic goitre. The first two patients were treated in London under the Mr. Selwyn Taylor and the other five in Cairo. Ains Shams
University Hospital, Cairo, Egypt, United Arab Republic
magnesium metabolism.
The normal magnesium content of the serum is 1-3-5 mg. per 100 ml., 20% being proteinbound and the remainder ionised. Dine and Lavietes 48 showed that in myxoedema all the magnesium was ionised while in hyperthyroidism up to half was protein-bound. Tapley 49 reported that administration of triiodothyronine to patients with myxoedema caused a rise in the urinary excretion of magnesium. It is likely that magnesium antagonises the effect of thyroxine on oxidation in the cells. Normally, energy released by oxidation in the cells is partly converted into heat and partly stored as energy-rich phosphate bonds in adenosine triphosphate. This coupling takes place in the mitochondria, and uncoupling results in a raised basal metabolic rate (B.M.R.). It can be produced by thyroxine and dinitrophenol. Feldott and Lardy 50 showed that, in vitro, thyroxine uncouples oxidative phosphorylation. Brody et al.51 showed that the uncoupling action of thyroxine was enhanced when the magnesium level fell below 12 micromoles per ml. and was r.eversed with magnesium levels above this. The 47. 48. 49. 50. 51.
Irie, M., Barrett, R. J. Endocrinology, 1962, 71, 277. Dine, R. F., Lavietes, P. H. J. clin. Invest. 1942, 21, 781. Tapley, D. F. J. biol. Chem. 1956, 222, 325. Feldott, G., Lardy, H. A. ibid. 1951, 192, 447. Brody, T. M., Hurwitz, R., Bain, J. A. Antibiot. and Chemother. 1954, 4, 864.
a
at
care
of
M. A. NEGUIB M.S.
Cairo
Medical Societies BRITISH ORTHOPÆDIC ASSOCIATION THE spring meeting of the British Orthopeadic Association was held in Sheffield on May 16-18. Mr. F. W. HOLDSWORTH, the President, was in the chair. We summarise below a few of the contributions. Mr. SIDNEY PAPPWORTH (Sheffield), in a clinical demonstration on subtrochanteric osteotomy as primary treatment of fractures of the femoral neck, said that nailing subcapital fractures of the femoral neck resulted in 25% of success only. For the past ten years he had combined reduction of the fracture with a McMurray type of osteotomy and fixation with a nail plate, the nail being introduced through the cut end of the bone. The osteotomy must be done as described by McMurray with the shaft displaced to support the femoral head. Then it did not matter whether the fracture united or the femoral head became avascular, and patients were still able to walk in comfort. Mr. J. R. KIRKUP (Bath) reported 84 patients treated similarly with a follow-up of six months to six years. All the osteotomies united; 39% had non-union, of the fracture; and 32% had avascular necrosis. Patients walked on the avascular heads without pain. The procedure was particularly indicated 52.
Wiswell, J. G., Braverman, M. G. Endocrinology, 1957, 61,
153.