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CHROMATOGRAPHIC SEPARATION OF CORTICOIDS ON A THIN LAYER OF SILICA GEL
81 ments to on
to be even greater in the relatively unsupported superficial veins. In this context the apparent protective effect of the inguinal ligament during coughing is noteworthy, though it is presumably absent during more prolonged straining.
explain this
would expect this effect
the basis of the
original hypothesis. First, the subjects may have forgotten, or be unaware, that their parents had varicose
Summary
veins.
Secondly, with increasing age, the valves present in this situation may have become incompetent and therefore indetectable by this technique. It is relevant that in about a third of subjects with " primary " varicose veins there is a negative family history (Nicholson 1927, de Takats and Quint 1930, Fig. 7-Radiograph in subject with gross varicose veins showing reflux Jensen 1932, Larson down] saphenous (S) and femoral and Smith 1943); that (F) veins. is to sav. varicose veins of this type can be acquired. On the other hand, 6 of these subjects were under 30 years of age, and 6 over 50, yet in each group only 3 had detectable valves. Thirdly, it can be suggested that a true familial tendency to varicose veins chanced not to have been expressed in the parents of the 6 valveless subjects. However, so far as they were aware, no other members of the family had varicose veins. Thus, while there is a clear association between the presence of these valves and the absence of a familial
A technique for the detection of competent venous valves above the saphenofemoral junction in the living subject is described. A significant relation between the apparent absence of these valves and the presence of a family history of varicose veins is demonstrated. In all subjects with varicose veins these valves were incompetent in the erect posture. A mechanism by which the inguinal ligament protects the leg veins from the pressure effects of coughing is described. We wish to thank Prof. G. J. Fraenkel for his support and interest, and Dr. A. C. Begg for the facilities offered by the X-ray department. ’
Mr. G. F. S. Spears carried out the statistical analysis. Mr. N. Miller and Mrs. D. McPhea gave valuable technical assistance. REFERENCES
Basmajian, J. V. (1952) Surg. Gynec. Obstet. 95, 537. De Takats, G., Quint, H. (1930) ibid. 50, 545. Dodd, H., Cockett, F. B. (1956) The Pathology and Surgery of the Veins of the Lower Limb. Edinburgh. Edwards, J. E., Edwards, E. A. (1940) Amer. Heart J. 19, 338. Eger, S. A., Casper, S. L. (1943) J. Amer. med. Ass. 123, 148. Wagner, F. B. (1949) Postgrad. Med. 6, 234. Hesse, E., Schaack, W. (1911) Virchows Arch. 205, 145. Höjensgård, I. C., Stürup, H. (1953) Acta physiol. scand. 27, 49. Jensen, D. R. (1932) Ann. Surg. 95, 738. Larson, R. A., Smith, F. L. (1943) Proc. Mayo Clin. 18, 400. McMurrich, J. P. (1906) Brit. med. J. ii, 1699. Nicholson, B. B. (1927) Arch. Surg., Chicago, 15, 351. Powell, T., Lynn, R. B. (1951) Surg. Gynec. Obstet. 92, 453. Smirk, F. H. (1936) Clin. Sci. 2, 317. Trendelenburg, F. (1891) Beitr. klin. Chir. 7, 195. Warwick, W. T. (1930) Lancet, ii, 1278. —
RELATION BETWEEN A DETECTABLE VALVE ABOVE THE SAPHENOFEMORAL JUNCTION, FAMILY HISTORY, AND VARICOSE VEINS
z
In both cases the valve was incompetent when the t In two cases the valve was incompetent when the
subject was subject was
erect. erect.
trend to varicose veins, it seems probable that the inherited defect is not confined to this set of valves alone. The fact that none of the 12 subjects examined with varicose veins had a valve above the saphenofemoral junction which was competent in the erect posture does reinforce the idea that these valves play a significant part in the development of internal-saphenous varices. It also lends direct support to the hypothesis of sequential valvular incompetence, particularly since in 2 of the subjects the calf varices were small, and treatment was not
being sought.
The observation that 4 subjects had valves which were competent while supine but incompetent when erect (when the hydrostatic pressure at rest in the femoral vein was some 30 mm. Hg higher) directly supports the anatomical evidence of Eger and Casper (1943) that the valvular defect in primary varicose veins is not damage to the cusps but dilatation of the valve ring. The pressures of up to 180 mm. Hg reached in the femoral vein in the erect posture, during the very moderate coughing employed by these subjects, can readily be imagined to lead to dilatation of the valve ring in this situation. One
Preliminary Communication CHROMATOGRAPHIC SEPARATION OF CORTICOIDS ON A THIN LAYER OF SILICA GEL THE earlier methods of fractionating of corticoids are too laborious for routine use in clinical laboratories. We have therefore devised a simple chromatographic technique suitable for analytical separation of corticoid mixtures. METHOD
Silica gel is prepared from ordinary waterglass by dilution with water in the ratio of 1/2 and precipitation with concentrated HCl, stirring vigorously until the pH becomes stable at 5. This crude preparation of silica gel is dried for three days at 80°C so that it acquires a granular consistence which can readily be rinsed. The dried crude silica is rinsed in a Buchner funnel with distilled water until the filtrate gives a negative reaction for chlorides. The rinsed silica is extracted with chloroform for eight hours in a Soxhlet extractor. The purified and dried preparation is then ground in a ball mill, and sifted to a grain size of 0.1-0.071 by means of a series of calibrated sieves. The moisture content is standardised by heating the silica gel thus obtained for forty-eight hours at 110°C, and the preparation is stored in a tight stoppered bottle. The technique of chromatography on thin layers of this
silica gel has proved successful:: 1.
Stárka, L., Malíková, J. J. Endocrin. 1961, 22,
215.
82 Silica gel is spread over the entire area of a 12 x 40 cm. glass plate, and divided into long strips by means of glass rods provided with rings 0-8 cm. wide, of polyethylene tubing. The distance between these rings depends on the required width of the silica-gel strips, and is usually 1.5 cm. The glass rods are drawn along the glass plate covered with silica gel to form the chromatographic strips. By adding silica gel to places where the layer is impaired, and by repeatedly moving the rods along, a continuous homogenous strip of silica gel can readily be obtained. The evaporate of the sample for chromatographic analysis is dissolved in enough chloroform-methanol 1/1 to allow for each silica-gel strip, 0-1-0-2 mg. of total corticoids with a maximum volume of 0-05 ml. The specimen is transferred by a micropipette to a distance of 10 cm. from the lower end of the strip: the diameter of the spot must not exceed 0-8 cm. The solvent is evaporated by an infra-red lamp. The chromatogram is developed in a glass vessel of 50 x 25 x 25 cm., into which a plate carrying the silica-gel strips is inserted at an angle of about 20° so that its lower edge lies in a petri dish. The mobile phase (a 5% solution of absolute ethanol in redistilled chloroform) is poured carefully into the petri dish through a long funnel, and the chamber is closed with a glass plate. The chromatogram is then developed for two hours. After the developed chromatogram has been dried under an infra-red lamp, the detection of corticoids is performed with an alkaline solution of tetrazolium blue 2; this is taken up from a pipette along the edge of the chromatographic strip. LABORATORY
To
These preliminary results suggest that the method will be successful for quantitative analyses. OTTO ADAMEC
Endocrinological Institute,
Lubochňa, Czechoslovakia
Ing. Chem. Prague JÁN MATIS Ing. Chem. Bratislava MARCEL GALVÁNEK Ing. Chem. Bratislava
New Inventions GAS-HEAT EXCHANGER FOR CARDIAC SURGERY THE extent to which it is desirable to use a pump, an oxygenator, and a heat-exchanger in open heart surgery is not yet determined. We therefore incorporated these three aids when we designed our apparatus for extracorporeal circulation. In this way the drawbacks of using an oxygenator and also a heat-exchanger separately are at least partly avoided. Others have made similar attempts.! We thought the simplest solution would be to make use of the common principle which underlies gas-and-heat exchange -namely, surface-enlarging of blood. With this in mind we
TESTS
separating capacity of the method described, standard preparations of pure corticoids were used: test
the
separation was very effective, and the individual spots were clearly defined. The mobility of individual steroids is expressed The
relative to that of substance F, the distance of which from the The average values of relative mobility start is taken as 1. from five chromatograms were: THF 0-43; THE 074; F 1-00; E 1-61; and s 2.00. The above values were well reproduced for individual batches of silica gel, the deviations never exceeding 0-02. The sensitivity of detection was about 1 }jLg. per sq. cm. CLINICAL APPLICATION
Satisfactory results were obtained when the above technique of chromatographic analysis was used for the separation of corticoids in urinary extracts from three The urine was hydrolysed with healthy subjects. and extracted with chloroform. The P-glucuronidase extract was purified by shaking with 0-1 N NaOH solution and with water, and then it was evaporated to dryness. On comparison with standard steroids processed in parallel, a THF spot, a very strong THE spot, substance F, and a trace of substance E were found in all urine samples. There were also one or two spots which so far have not been identified.
interesting steroid spectrum was obtained in a patient with congenital hyperplasia of the adrenal cortex, An
a small amount of THE substance but a a corticoid the spot of which appeared of large in the region between substances F and E. This is most probably THS, which corresponds to the abnormal steroid produced in this disease.
who excreted amount
2.
Bush, I. E., Willoughby, M. Biochem. J. 1957, 67, 689.
The gas-heat exchanger. Above: vertical median section. Below: horizontal section through cooling ribs.
studied the basic construction of a disc oxygenator. The least distance between the discs is determined by the need to prevent filming and foaming between adjacent discs above the level of the blood in the apparatus. Below this level discs are, of course, the same distance apart and this accounts for the rather high priming volume required for disc oxygenators. We interposed cooling ribs into the spaces between the discs below the blood-level, and this gave a combined surface which compares favourably with that of conventional heatexchangers. In this way the function of heat exchange is added to the disc oxygenator, and at the same time its priming volume is reduced (even taking into account the fact that convoluted discs cannot be used). The mechanical trauma to the blood caused by gas and heat exchange is also reduced because one surface-enlarging serves both functions. The figure shows schematically the construction of our gas-heat exchanger. Essentially it is a stainless-steel block with alterr.ating grooves above (where blood circulates and discs 1.
Osborn, J. J., Bramson, M. L., Gerbode, F. J. thorac. Surg. 1960, 39, 427
to be even greater in the relatively unsupported superficial veins. In this context the apparent protective effect of the inguinal ligament during coughing is noteworthy, though it is presumably absent during more prolonged straining.
explain this
would expect this effect
the basis of the
original hypothesis. First, the subjects may have forgotten, or be unaware, that their parents had varicose
Summary
veins.
Secondly, with increasing age, the valves present in this situation may have become incompetent and therefore indetectable by this technique. It is relevant that in about a third of subjects with " primary " varicose veins there is a negative family history (Nicholson 1927, de Takats and Quint 1930, Fig. 7-Radiograph in subject with gross varicose veins showing reflux Jensen 1932, Larson down] saphenous (S) and femoral and Smith 1943); that (F) veins. is to sav. varicose veins of this type can be acquired. On the other hand, 6 of these subjects were under 30 years of age, and 6 over 50, yet in each group only 3 had detectable valves. Thirdly, it can be suggested that a true familial tendency to varicose veins chanced not to have been expressed in the parents of the 6 valveless subjects. However, so far as they were aware, no other members of the family had varicose veins. Thus, while there is a clear association between the presence of these valves and the absence of a familial
A technique for the detection of competent venous valves above the saphenofemoral junction in the living subject is described. A significant relation between the apparent absence of these valves and the presence of a family history of varicose veins is demonstrated. In all subjects with varicose veins these valves were incompetent in the erect posture. A mechanism by which the inguinal ligament protects the leg veins from the pressure effects of coughing is described. We wish to thank Prof. G. J. Fraenkel for his support and interest, and Dr. A. C. Begg for the facilities offered by the X-ray department. ’
Mr. G. F. S. Spears carried out the statistical analysis. Mr. N. Miller and Mrs. D. McPhea gave valuable technical assistance. REFERENCES
Basmajian, J. V. (1952) Surg. Gynec. Obstet. 95, 537. De Takats, G., Quint, H. (1930) ibid. 50, 545. Dodd, H., Cockett, F. B. (1956) The Pathology and Surgery of the Veins of the Lower Limb. Edinburgh. Edwards, J. E., Edwards, E. A. (1940) Amer. Heart J. 19, 338. Eger, S. A., Casper, S. L. (1943) J. Amer. med. Ass. 123, 148. Wagner, F. B. (1949) Postgrad. Med. 6, 234. Hesse, E., Schaack, W. (1911) Virchows Arch. 205, 145. Höjensgård, I. C., Stürup, H. (1953) Acta physiol. scand. 27, 49. Jensen, D. R. (1932) Ann. Surg. 95, 738. Larson, R. A., Smith, F. L. (1943) Proc. Mayo Clin. 18, 400. McMurrich, J. P. (1906) Brit. med. J. ii, 1699. Nicholson, B. B. (1927) Arch. Surg., Chicago, 15, 351. Powell, T., Lynn, R. B. (1951) Surg. Gynec. Obstet. 92, 453. Smirk, F. H. (1936) Clin. Sci. 2, 317. Trendelenburg, F. (1891) Beitr. klin. Chir. 7, 195. Warwick, W. T. (1930) Lancet, ii, 1278. —
RELATION BETWEEN A DETECTABLE VALVE ABOVE THE SAPHENOFEMORAL JUNCTION, FAMILY HISTORY, AND VARICOSE VEINS
z
In both cases the valve was incompetent when the t In two cases the valve was incompetent when the
subject was subject was
erect. erect.
trend to varicose veins, it seems probable that the inherited defect is not confined to this set of valves alone. The fact that none of the 12 subjects examined with varicose veins had a valve above the saphenofemoral junction which was competent in the erect posture does reinforce the idea that these valves play a significant part in the development of internal-saphenous varices. It also lends direct support to the hypothesis of sequential valvular incompetence, particularly since in 2 of the subjects the calf varices were small, and treatment was not
being sought.
The observation that 4 subjects had valves which were competent while supine but incompetent when erect (when the hydrostatic pressure at rest in the femoral vein was some 30 mm. Hg higher) directly supports the anatomical evidence of Eger and Casper (1943) that the valvular defect in primary varicose veins is not damage to the cusps but dilatation of the valve ring. The pressures of up to 180 mm. Hg reached in the femoral vein in the erect posture, during the very moderate coughing employed by these subjects, can readily be imagined to lead to dilatation of the valve ring in this situation. One
Preliminary Communication CHROMATOGRAPHIC SEPARATION OF CORTICOIDS ON A THIN LAYER OF SILICA GEL THE earlier methods of fractionating of corticoids are too laborious for routine use in clinical laboratories. We have therefore devised a simple chromatographic technique suitable for analytical separation of corticoid mixtures. METHOD
Silica gel is prepared from ordinary waterglass by dilution with water in the ratio of 1/2 and precipitation with concentrated HCl, stirring vigorously until the pH becomes stable at 5. This crude preparation of silica gel is dried for three days at 80°C so that it acquires a granular consistence which can readily be rinsed. The dried crude silica is rinsed in a Buchner funnel with distilled water until the filtrate gives a negative reaction for chlorides. The rinsed silica is extracted with chloroform for eight hours in a Soxhlet extractor. The purified and dried preparation is then ground in a ball mill, and sifted to a grain size of 0.1-0.071 by means of a series of calibrated sieves. The moisture content is standardised by heating the silica gel thus obtained for forty-eight hours at 110°C, and the preparation is stored in a tight stoppered bottle. The technique of chromatography on thin layers of this
silica gel has proved successful:: 1.
Stárka, L., Malíková, J. J. Endocrin. 1961, 22,
215.
82 Silica gel is spread over the entire area of a 12 x 40 cm. glass plate, and divided into long strips by means of glass rods provided with rings 0-8 cm. wide, of polyethylene tubing. The distance between these rings depends on the required width of the silica-gel strips, and is usually 1.5 cm. The glass rods are drawn along the glass plate covered with silica gel to form the chromatographic strips. By adding silica gel to places where the layer is impaired, and by repeatedly moving the rods along, a continuous homogenous strip of silica gel can readily be obtained. The evaporate of the sample for chromatographic analysis is dissolved in enough chloroform-methanol 1/1 to allow for each silica-gel strip, 0-1-0-2 mg. of total corticoids with a maximum volume of 0-05 ml. The specimen is transferred by a micropipette to a distance of 10 cm. from the lower end of the strip: the diameter of the spot must not exceed 0-8 cm. The solvent is evaporated by an infra-red lamp. The chromatogram is developed in a glass vessel of 50 x 25 x 25 cm., into which a plate carrying the silica-gel strips is inserted at an angle of about 20° so that its lower edge lies in a petri dish. The mobile phase (a 5% solution of absolute ethanol in redistilled chloroform) is poured carefully into the petri dish through a long funnel, and the chamber is closed with a glass plate. The chromatogram is then developed for two hours. After the developed chromatogram has been dried under an infra-red lamp, the detection of corticoids is performed with an alkaline solution of tetrazolium blue 2; this is taken up from a pipette along the edge of the chromatographic strip. LABORATORY
To
These preliminary results suggest that the method will be successful for quantitative analyses. OTTO ADAMEC
Endocrinological Institute,
Lubochňa, Czechoslovakia
Ing. Chem. Prague JÁN MATIS Ing. Chem. Bratislava MARCEL GALVÁNEK Ing. Chem. Bratislava
New Inventions GAS-HEAT EXCHANGER FOR CARDIAC SURGERY THE extent to which it is desirable to use a pump, an oxygenator, and a heat-exchanger in open heart surgery is not yet determined. We therefore incorporated these three aids when we designed our apparatus for extracorporeal circulation. In this way the drawbacks of using an oxygenator and also a heat-exchanger separately are at least partly avoided. Others have made similar attempts.! We thought the simplest solution would be to make use of the common principle which underlies gas-and-heat exchange -namely, surface-enlarging of blood. With this in mind we
TESTS
separating capacity of the method described, standard preparations of pure corticoids were used: test
the
separation was very effective, and the individual spots were clearly defined. The mobility of individual steroids is expressed The
relative to that of substance F, the distance of which from the The average values of relative mobility start is taken as 1. from five chromatograms were: THF 0-43; THE 074; F 1-00; E 1-61; and s 2.00. The above values were well reproduced for individual batches of silica gel, the deviations never exceeding 0-02. The sensitivity of detection was about 1 }jLg. per sq. cm. CLINICAL APPLICATION
Satisfactory results were obtained when the above technique of chromatographic analysis was used for the separation of corticoids in urinary extracts from three The urine was hydrolysed with healthy subjects. and extracted with chloroform. The P-glucuronidase extract was purified by shaking with 0-1 N NaOH solution and with water, and then it was evaporated to dryness. On comparison with standard steroids processed in parallel, a THF spot, a very strong THE spot, substance F, and a trace of substance E were found in all urine samples. There were also one or two spots which so far have not been identified.
interesting steroid spectrum was obtained in a patient with congenital hyperplasia of the adrenal cortex, An
a small amount of THE substance but a a corticoid the spot of which appeared of large in the region between substances F and E. This is most probably THS, which corresponds to the abnormal steroid produced in this disease.
who excreted amount
2.
Bush, I. E., Willoughby, M. Biochem. J. 1957, 67, 689.
The gas-heat exchanger. Above: vertical median section. Below: horizontal section through cooling ribs.
studied the basic construction of a disc oxygenator. The least distance between the discs is determined by the need to prevent filming and foaming between adjacent discs above the level of the blood in the apparatus. Below this level discs are, of course, the same distance apart and this accounts for the rather high priming volume required for disc oxygenators. We interposed cooling ribs into the spaces between the discs below the blood-level, and this gave a combined surface which compares favourably with that of conventional heatexchangers. In this way the function of heat exchange is added to the disc oxygenator, and at the same time its priming volume is reduced (even taking into account the fact that convoluted discs cannot be used). The mechanical trauma to the blood caused by gas and heat exchange is also reduced because one surface-enlarging serves both functions. The figure shows schematically the construction of our gas-heat exchanger. Essentially it is a stainless-steel block with alterr.ating grooves above (where blood circulates and discs 1.
Osborn, J. J., Bramson, M. L., Gerbode, F. J. thorac. Surg. 1960, 39, 427