- Email: [email protected]
MEASUREMENT OF LIVER-IRON CONCENTRATION IN NEEDLE-BIOPSY SPECIMENS
100
of the colon, but we believe that the results of our present survey indicate that further exploration of the part played by bacteria may well be very rewarding. This work was supported financially by the Cancer Research Campaign M. J. H., V. C. A., and J. S. C.), the Wellcome Foundation B. S. D.. and the British Nutrition Foundation (G- M. H.. We are greatly indebted to colleagues who arranged for the collection and dispatch of the faecal samples: Dr. R. Blowers (Uganda . Dr. P. Brachman (Atlanta), Dr. T. Shigematsu (Yonaga, Prof. S. Baker (Vellore), and Dr. G. Collee (Edinburgh’;and to Prof. Richard Doll for much helpful advice. REFERENCES
1. Doll, R., Paync, P., Waterhouse, J. (editors). Cancer Incidence in Five Continents. Berlin, 1966. 2. Doll. R. Natn, Cancer Inst. Monogr. 1967, 25, 173. 3. Doll, R. Br. J. Cancer, 1969, 23, 1. 4. Buell, P., Dunn, J. E. Cancer, 1965, 18, 656. 5. Wynder, E. L., Kajitani, T., Ushikawa, S., Dodo, H., Jakano, A. ibid. 1969, 23, 1210. 6. Wynder, E. L., Shigematsu, T. ibid. 1967, 20, 1520. 7. Gregor, O., Toman, R., Prusova, F. Gut, 1969, 10, 1031. 8. Ghiron, V. Int. Cancer Congr., 1939, p. 116. 9. Badger, G. M., Cook, J. W., Hewett, C. L., Kennaway, E. L., Kennaway, N. M., Martin, R. H., Robinson, A. M. Proc. R. Soc. B, 1940, 129, 439. 10. Cook, J. W., Kennaway, E. L., Kennaway, N. M. Nature, 1940, 145, 627. 11. Lacassagne, A., Buu-Hoi, N. P., Zajdela, F. ibid. 1966, 209, 1026. 12. Lacassagne, A., Buu-Hoi, N. P., Zajdela, F. ibid. 1961, 190, 1007. 13. Inhoffen, H. H. Prog. org. Chem. 1953, 2, 131. 14. Cook, J. W., Haselwood, G. D. A. Chemy Ind. Rev. 1933, 11, 758. 15. Druckrey, H., Richter, R., Vierthaler, R. Klin. Wschr. 1941, 26, 781. 16. Hoffmann, K. Zbl. Bakt. 1 Abt. Orig. 1964, 192, 500. 17. Hill, M. J. J. Path. (in the press). 18. Crowther, J. S. J. appl. Bact. (in the press). 19. Drasar, B. S. J. Path. Bact. 1967, 94, 417. 20. Drasar, B. S., Crowther, J. S. Tech. Ser. Soc. appl. Bact. 1970, no. 5. 21. Hill, M. J., Aries, V. C. J. Path. (in the press). 22. Crowther, J. S. J. med. Microbiol. (in the press). 23. Ellis, F. R., Mumford, P. Proc. Nutr. Soc. 1967, 26, 205. 24. Dietschy, J. M. Gastroenterology, 1969, 57, 461. 25. Danielsson, H. Acta physiol. scand. 1960, 48, 364. 26. Aries, V. C., Crowther, J. S., Drasar, B. S., Hill, M. J. Gut, 1969, 10, 575. 27. Aries, V. C., Hill, M. J. Biochem. J. 1970, 119, 37P. 28. Hill, M. J. Marie Curie Foundation workshop conference on Some Implication of Steroid Hormones in Cancer (in the press). 29. Hawksworth, G. M., Hill, M. J. Biochem. J. (in the press). 30. Magec, P. N., Barnes, J. M. Adv. Cancer Res. 1967, 10, 163.
MEASUREMENT OF LIVER-IRON CONCENTRATION IN NEEDLE-BIOPSY SPECIMENS MICHAEL BARRY SHEILA SHERLOCK
Department of Medicine, Royal Free Hospital, London W.C.1
Summary
simple chemical method (with bathophenanthroline sulphonate) was used to
A
determine total liver-iron concentration in needlebiopsy specimens from patients with various ironloading disorders and with chronic liver disease. Diethylenetriamine penta-acetic acid (D.T.P.A.) chelatable iron, which provides an indirect quantitative estimate of total body iron stores, was also determined in most of the cases. There was a high degree of correlation between liver-iron concentration and D.T.P.A.chelatable iron. The coefficient of variation for iron concentration in duplicate specimens from the same liver was 8.6% and 7.1% in control material and patients with liver disease respectively. Determination of total liver-iron concentration in needle-biopsy specimens is a simple and generally practicable method
for
measuring tissue iron stores, the result closely reflecting total body-storage iron. The method does not measure non-hæm iron selectively, however, and may therefore be imprecise at iron-depletion levels. Introduction LACK of a simple method for direct measurement of tissue iron stores has for long hampered the clinical investigation of iron metabolism. Of the indirect techniques, determination of mobilisable storage iron by frequent venesection is the most reliable, 1-3 and has been used to calibrate more practicable methods using
chelating agents. 4-7 Direct chemical estimation of liver-iron concentration is a logical extension of the widely used histochemical method for assessing the stores, but has seldom been done on biopsy tissue."’" Although nonhaem iron concentration can be measured in liverbiopsy specimens,8,11 theoretical consideration suggested that the contribution of haemoglobin-iron to the total iron content of such specimens would be small, and insignificant in the presence of iron excess. Furthermore, a method for determining total liveriron concentration might be very simple, whereas for non-hxm iron special extraction procedures are
required. We have used a simple chemical method to determine total liver-iron concentration in needle-biopsy specimens. The findings in patients with idiopathic and secondary hxmochronaatosis, and in those with other disorders, were closely correlated with estimates of total body-storage iron based on a chelation test using diethylenetriamine penta-acetic acid (D.T.P.A.).’ Methods Percutaneous liver sampling was done with a Menghini needle of internal diameter 1-9 mm. The specimen was expelled onto ashless (’ Whatman 42 ’) filter paper, separated from extraneous blood, and gently rinsed with a few drops of saline solution from the biopsy needle. Part of the specimen was fixed in iron-free formol-saline for routine histological examination; the remainder was transferred to a small iron-freePyrex ’ vessel, and the dry weight was determined after oven drying at 120°C to constant weight. It was then ashed with 015 ml. of a 1/1 (v/v) mixture of concentrated sulphuric and nitric acids in a 30 ml. Kjeldahl flask. The technique, modified from a procedure for ashing urine,’is suitable for specimens of up to 20 mg. dry weight. A clear, colourless residue was obtained after heating for 5-10 minutes over a low flame. A blank was treated similarly. When the residue had cooled, 15 ml. of ion-free water was pipetted into the flask. After tightly sealing its mouth with’Parafilm ’, the flask was inverted several times and the contents thoroughly mixed. A sample from each flask was estimated for iron using bathophenanthroline
sulphonate. 13 Histological examination of the specimen included assessment of the stainable iron content of the parenchymal cells using Perls’ technique, grade 0 representing absence of TABLE
I—MEAN(&sfgr;±S.E.M.)
VALUES IN CONTROLS
101 siderosis and grades 1-4 representing increasing degrees of iron deposition. 14 D.T.p.A.-chelatable body iron was determined using the chelation test previously described.7 Predicted total bodystorage iron (g.)= D.T.P.A.-chelatable iron (mg.) x 0-46, the standard deviation for such estimates being 112 g. in
patients with
iron
excess
whose
stores were
measured
by
venesection.7
Results
The findings in controls are shown in table i. Most of the specimens were obtained by post-mortem needle
TABLE II-FINDINGS IN PATIENTS WITH LIVER DISEASE AND VARIOUS IRON-LOADING DISORDERS
102 Additional quantitative measurecreased.10,11,15,1616 ments are desirable to confirm the presence of iron excess and to form a basis for rational management. Although acceptably precise estimates of body-iron stores are now possible with certain chelation tests, such measurements are essentially indirect, and the techniques, which entail radioisotope administration, are somewhat specialised. Liver biopsy is commonly done in patients thought to have iron excess, and the determination of iron concentration in such specimens, using the simple chemical procedure described here, has been found useful and should prove generally
practicable.
Relation between liver-iron concentration and D.T.P.A.-chelatable iron.
y=76-24x-35-98. (r=0963.) Predicted total body-storage iron (g.) D.T. P.A.-chelatable iron (mg.) x0.46.
=
sampling from patients who had died in hospital from myocardial infarction or cerebrovascular accident. None had a history of recent blood-loss, blood-transfusion, or surgical operation, and none had had liver disease. The average dry weight of the specimens was 9-4 mg. (range 41-193 mg.). In eight cases two specimens were examined, each from different parts of the liver; the standard deviation for a single specimen was 6-5 µg. iron per 100 mg. dry weight, which gave a coefficient of variation of 86°. The findings in patients with various forms of iron overload and in those with chronic liver disease are shown in table 11. The average dry weight of these specimens was 54 mg. (range 22-120 mg.). In 12 cases duplicate specimens were examined; the standard deviation for a single specimen was 123 µg. iron per 100 mg. dry weight (the mean iron concentration was 1738 µg. per 100 mg. dry weight), corresponding to a coefficient of variation of 7.1%. D.T.P.A.-chelatable iron was also determined in most of the cases listed in table 11, enabling total bodystorage iron to be predicted. As shown in the accompanying figure (in which logarithmic scales have been used for convenience) there was a highly significant correlation between liver-iron concentration and D.T.P.A.-chelatable iron (or predicted total body storage iron) (r=0.963; P "0001). The regression intercept did not differ significantly from the origin (P>0.6). The standard deviation for single estimates of total body-iron stores based on liver-iron concentration was 1 45 g. Discussion
Histochemical examination of liver-biopsy specimens is commonly used to assess the size of the iron stores and is valuable for screening purposes. The findings, however, are not always easy to interpret since appreciable amounts of stainable iron may be present when tissue-iron levels are normal or only slightly in-
The method measures total (as opposed to nonhaem) iron concentration, and the result therefore includes the hxmoglobin-iron content of the specimen. Assuming that liver tissue contains about 8% blood," the contribution due to hæmoglobin-iron may amount to 12 µg. per 100 mg. dry weight. This quantity is unimportant in the presence of iron excess but may be highly significant when there is storage iron depletion. Although we did not investigate liver biopsies from patients with chronic iron-deficiency anaemia, Weinfeld found that non-hæm iron in such patients was 2-10 µg. per 100 mg. dry weight, which would be expected to correspond to total iron concentrations of less than 25 µg. per 100 mg. dry weight. 11
The control observations accord well with pre-
viously published figures for normal human storage iron in adult liver obtained at necropsy.18 They also conform closely with values for non-haem iron in tissue obtained by excision biopsy 8,9and by aspiration needle-biopsy 10,11 from carefully selected controls undergoing surgery for uncomplicated biliary or peptic-ulcer disease. The failure to demonstrate a sex difference in our controls requires comment, however, since Weinfelda found that mean hepatic storage iron in postmenopausal women was significantly lower than in control men (though higher than in menstruating women). The explanation probably resides in the more advanced age of the females in our series (mean age 77, compared with mean age 60 in Weinfeld’s series). Close agreement between duplicate biopsies from the same liver has been previously demonstrated in controls,11 and it was reassuring to obtain similar concordance in specimens from patients with chronic liver disease, both with and without iron excess. The distribution of fat, fibrosis, and siderosis is often patchy, and greater sample variation might have been expected. However, the organ-distribution of storage iron may not be so uniform. Thus, in idiopathic hxmochromatosis deposition in hepatic parenchymal cells predominates and the spleen and bone-marrow are largely spared, whereas in hsemolytic states and transfusional overload the reticuloendothelial system bears the initial impact. Nevertheless, reticuloendothelial deposits are thought to be gradually redistributed to parenchymal stores in time, and the liver, which contains both components, has been regarded as generally most representative of total body iron content.19 Discrepancies between liver and bone-marrow do seem to be more striking at lower storage iron concentrations,and over a wide range of values a good correlation between the two sites was found." This is reflected here, in patients with various forms of iron
103
by the close correlation between liver iron concentration and D.T.p.A.-chelatable iron, for the latter has been shown to be highly correlated with total body storage iron as measured by venesection.7 Although our findings suggest that liver-iron concentration may be less precise than the chelation test for measuring total body-iron stores, the difference between the two methods would seem to be relatively excess,
FATE OF KIDNEY ALLOGRAFTS FROM CADAVERS WHOSE LIVERS WERE ALSO
TRANSPLANTED ALAN J. L. HART W. A. B. SMELLIE R. Y. CALNE
Department of Surgery, University of Cambridge
slight. A histogenetic relationship between iron excess and hepatic fibrosis has long been assumed in idiopathic and secondary hasmochromatosis, but experimental evidence in support of this concept has only lately been obtained.21 The quantitative aspects of the association have yet to be defined. Most patients with idiopathic hxmochromatosis seen at this hospital have iron loads of 10-15 g. (corresponding to liver-iron concentrations of 1-6-2-6% dry weight according to our data); fibrosis is usually heavy and nodules often present. This
Where both liver and kidneys are removed from a cadaver for transplantathe liver takes priority, so that there is a delay tion, before individual perfusion of the kidney can be started. However, experience with twenty renal allografts taken from 11 cadavers which were primarily liver donors suggests that kidneys from this source can be confidently used in renal transplant operations. Eighteen of these kidneys functioned immediately, and 7 weeks to 21 months after operation, fifteen of these are still functioning satisfactorily. All donors were
accords with the observation in Bantu siderosis that heavy fibrosis or cirrhosis is frequent when liver iron concentration exceeds 2% dry weight.22, 233 However, the actual amount of iron present may not be the only factor concerned in the pathogenesis of tissue injury, for only one of the four young patients with idiopathic haemochromatosis studied by Charlton et al.24 had appreciable fibrosis, though all had liver-iron contents exceeding 2%. Moreover, special factors seem to operate in pure transfusional overload (e.g., in aplastic anaemia), where fibrosis may be trivial despite iron excess of massive proportions (vide case 9). This has suggested that transfused hxmoglobin-iron may be relatively bland, perhaps because it is initially processed by the reticuloendothelial system, whereas excess iron accumulated via the gut is fibrogenic in much smaller amounts.25 Further understanding of the role of iron in the pathogenesis of liver damage must partly depend on correlating histological and chemical data in patients with iron-loading states, and in this respect valuable information may be obtained from prospective clinical studies with serial biopsies in patients with various forms of refractory anaemia.
respirator
We thank Dr. Peter Scheuer for the liver-biopsy interpretations, Dr. Michael Wills for the serum-iron estimations, and the many physicians who have referred patients to us.
Requests
for
reprints should be addressed to
S. S.
REFERENCES 1.
2. 3. 4.
Haskins, D., Stevens, A. R., Finch, S., Finch, C. A. J. clin. Invest. 1952, 31, 543. Pritchard, J. A., Mason, R. A. J. Am. med. Ass. 1964, 190, 897. Hynes, M. J. clin. Path. 1949, 2, 99. Balcerzak, S. P., Westerman, M. P., Heinle, E. W., Taylor, F. H.
Ann. intern. Med. 1968, 68, 518. Smith, P. M., Lestas, A. N., Miller, J. G. P., Dymock, I. W., Pitcher, C. S., Williams, R. Lancet, 1969, ii, 402. 6. Barry, M., Cartei, G. C., Sherlock, S. Gut, 1969, 10, 697. 7. Barry, M., Cartei, G. C., Sherlock, S. ibid. 1970, 11, 891. 8. Weinfeld, A. Acta med. scand. 1964, 177, suppl. 427. 9. Hallberg, L., Hedenberg, L., Weinfeld, A. Scand. J. Hœmat. 1966, 3, 85. 10. Weinfeld, A., Lundin, P., Lundvall, O. J. clin. Path. 1968, 21, 35. 11. Lundvall, O., Weinfeld, A., Lundin, P. Acta med. scand. 1969, 185, 5.
259. 12.
Frey, W. G., Gardner, M. H., Pillsbury, J. A. J. Lab. clin. Med. 1968, 72, 52. 13. Barry, M. J. clin. Path. 1968, 21, 166.
Summary
cases.
Introduction
WE describe here the fate of twenty renal allografts taken from 11 cadavers which had also been used for liver transplantation. These kidneys were obtained under conditions which differ from those in cases where kidneys are the primary donor organ, since removal of the liver must take precedence because of its low tolerance to ischxmia. Methods In all cases the donors had irreversible brain damage, and ventilation was maintained artificially. Doctors independent of the transplant team decided in each case to stop mechanical respiration once irreversible brain death had been diagnosed. They informed the transplant team of this decision so that preparations could be made for a livergrafting operation. After mechanical respiration had been stopped, the surgeons waited until the circulation had ceased (as determined by doctors independent of the transplant team) before starting to remove the liver. Where the kidneys alone are to be transplanted, we remove organs as quickly as possible and perfuse them by the Gelin technique,l2with refrigerated Rheomacrodex until the renal-vein effluent is clear and following this with 200-300 ml. of a mixture of 10% fructose and 1-4% sodium bicarbonate. The kidneys are then packed in ice and placed in a thermos flask until the time of implantation in the recipient. However, where the liver is to be removed, the kidneys remain in situ while the liver is perfused via the portal vein with Hartmann’s solution, followed by a mixture of plasma, bicarbonate, and dextrose. The last two cases have also been perfused via the aorta in order that the hepatic artery may be perfused and also to acceler-
14.
Scheuer, P. J., Williams, R., Muir,
A. R.
J. Path.
Bact.
1962, 84,
53.
15. Finch, C. A., Bothwell, T. H. Archs intern. Med. 1961, 107, 807. 16. Morgan, E. H., Walters, M. N. J. clin. Path. 1963, 16, 101. 17. Scott, E. M., McCoy, R. H. Archs Biochem. 1944, 5, 349. 18. Powell, L. W. Australas. Ann. Med. 1966, 15, 110. 19. Bothwell, T. H., Finch, C. A. Iron Metabolism. London, 1962. 20. Gale, E., Torrance, J., Bothwell, T. H. J. clin. Invest. 1963, 42, 1076. 21. Lisboa, P. E. 5th Meeting of the European Association for the Study of the Liver, 1970; abstracts, p. 29. 22. Bothwell, T. H., Bradlow, B. A. Archs Path. 1960, 70, 279. 23. Bothwell, T. H., Isaacson, C. Br. med. J. 1962, i, 522. 24. Charlton, R. W., Abrahams, C., Bothwell, T. H. Archs Path. 1967, 83, 132. 25. Cappell, D. F., Hutchison, H. E., Jowett, M. J. Path. Bact. 1957, 74, 245.
of the colon, but we believe that the results of our present survey indicate that further exploration of the part played by bacteria may well be very rewarding. This work was supported financially by the Cancer Research Campaign M. J. H., V. C. A., and J. S. C.), the Wellcome Foundation B. S. D.. and the British Nutrition Foundation (G- M. H.. We are greatly indebted to colleagues who arranged for the collection and dispatch of the faecal samples: Dr. R. Blowers (Uganda . Dr. P. Brachman (Atlanta), Dr. T. Shigematsu (Yonaga, Prof. S. Baker (Vellore), and Dr. G. Collee (Edinburgh’;and to Prof. Richard Doll for much helpful advice. REFERENCES
1. Doll, R., Paync, P., Waterhouse, J. (editors). Cancer Incidence in Five Continents. Berlin, 1966. 2. Doll. R. Natn, Cancer Inst. Monogr. 1967, 25, 173. 3. Doll, R. Br. J. Cancer, 1969, 23, 1. 4. Buell, P., Dunn, J. E. Cancer, 1965, 18, 656. 5. Wynder, E. L., Kajitani, T., Ushikawa, S., Dodo, H., Jakano, A. ibid. 1969, 23, 1210. 6. Wynder, E. L., Shigematsu, T. ibid. 1967, 20, 1520. 7. Gregor, O., Toman, R., Prusova, F. Gut, 1969, 10, 1031. 8. Ghiron, V. Int. Cancer Congr., 1939, p. 116. 9. Badger, G. M., Cook, J. W., Hewett, C. L., Kennaway, E. L., Kennaway, N. M., Martin, R. H., Robinson, A. M. Proc. R. Soc. B, 1940, 129, 439. 10. Cook, J. W., Kennaway, E. L., Kennaway, N. M. Nature, 1940, 145, 627. 11. Lacassagne, A., Buu-Hoi, N. P., Zajdela, F. ibid. 1966, 209, 1026. 12. Lacassagne, A., Buu-Hoi, N. P., Zajdela, F. ibid. 1961, 190, 1007. 13. Inhoffen, H. H. Prog. org. Chem. 1953, 2, 131. 14. Cook, J. W., Haselwood, G. D. A. Chemy Ind. Rev. 1933, 11, 758. 15. Druckrey, H., Richter, R., Vierthaler, R. Klin. Wschr. 1941, 26, 781. 16. Hoffmann, K. Zbl. Bakt. 1 Abt. Orig. 1964, 192, 500. 17. Hill, M. J. J. Path. (in the press). 18. Crowther, J. S. J. appl. Bact. (in the press). 19. Drasar, B. S. J. Path. Bact. 1967, 94, 417. 20. Drasar, B. S., Crowther, J. S. Tech. Ser. Soc. appl. Bact. 1970, no. 5. 21. Hill, M. J., Aries, V. C. J. Path. (in the press). 22. Crowther, J. S. J. med. Microbiol. (in the press). 23. Ellis, F. R., Mumford, P. Proc. Nutr. Soc. 1967, 26, 205. 24. Dietschy, J. M. Gastroenterology, 1969, 57, 461. 25. Danielsson, H. Acta physiol. scand. 1960, 48, 364. 26. Aries, V. C., Crowther, J. S., Drasar, B. S., Hill, M. J. Gut, 1969, 10, 575. 27. Aries, V. C., Hill, M. J. Biochem. J. 1970, 119, 37P. 28. Hill, M. J. Marie Curie Foundation workshop conference on Some Implication of Steroid Hormones in Cancer (in the press). 29. Hawksworth, G. M., Hill, M. J. Biochem. J. (in the press). 30. Magec, P. N., Barnes, J. M. Adv. Cancer Res. 1967, 10, 163.
MEASUREMENT OF LIVER-IRON CONCENTRATION IN NEEDLE-BIOPSY SPECIMENS MICHAEL BARRY SHEILA SHERLOCK
Department of Medicine, Royal Free Hospital, London W.C.1
Summary
simple chemical method (with bathophenanthroline sulphonate) was used to
A
determine total liver-iron concentration in needlebiopsy specimens from patients with various ironloading disorders and with chronic liver disease. Diethylenetriamine penta-acetic acid (D.T.P.A.) chelatable iron, which provides an indirect quantitative estimate of total body iron stores, was also determined in most of the cases. There was a high degree of correlation between liver-iron concentration and D.T.P.A.chelatable iron. The coefficient of variation for iron concentration in duplicate specimens from the same liver was 8.6% and 7.1% in control material and patients with liver disease respectively. Determination of total liver-iron concentration in needle-biopsy specimens is a simple and generally practicable method
for
measuring tissue iron stores, the result closely reflecting total body-storage iron. The method does not measure non-hæm iron selectively, however, and may therefore be imprecise at iron-depletion levels. Introduction LACK of a simple method for direct measurement of tissue iron stores has for long hampered the clinical investigation of iron metabolism. Of the indirect techniques, determination of mobilisable storage iron by frequent venesection is the most reliable, 1-3 and has been used to calibrate more practicable methods using
chelating agents. 4-7 Direct chemical estimation of liver-iron concentration is a logical extension of the widely used histochemical method for assessing the stores, but has seldom been done on biopsy tissue."’" Although nonhaem iron concentration can be measured in liverbiopsy specimens,8,11 theoretical consideration suggested that the contribution of haemoglobin-iron to the total iron content of such specimens would be small, and insignificant in the presence of iron excess. Furthermore, a method for determining total liveriron concentration might be very simple, whereas for non-hxm iron special extraction procedures are
required. We have used a simple chemical method to determine total liver-iron concentration in needle-biopsy specimens. The findings in patients with idiopathic and secondary hxmochronaatosis, and in those with other disorders, were closely correlated with estimates of total body-storage iron based on a chelation test using diethylenetriamine penta-acetic acid (D.T.P.A.).’ Methods Percutaneous liver sampling was done with a Menghini needle of internal diameter 1-9 mm. The specimen was expelled onto ashless (’ Whatman 42 ’) filter paper, separated from extraneous blood, and gently rinsed with a few drops of saline solution from the biopsy needle. Part of the specimen was fixed in iron-free formol-saline for routine histological examination; the remainder was transferred to a small iron-freePyrex ’ vessel, and the dry weight was determined after oven drying at 120°C to constant weight. It was then ashed with 015 ml. of a 1/1 (v/v) mixture of concentrated sulphuric and nitric acids in a 30 ml. Kjeldahl flask. The technique, modified from a procedure for ashing urine,’is suitable for specimens of up to 20 mg. dry weight. A clear, colourless residue was obtained after heating for 5-10 minutes over a low flame. A blank was treated similarly. When the residue had cooled, 15 ml. of ion-free water was pipetted into the flask. After tightly sealing its mouth with’Parafilm ’, the flask was inverted several times and the contents thoroughly mixed. A sample from each flask was estimated for iron using bathophenanthroline
sulphonate. 13 Histological examination of the specimen included assessment of the stainable iron content of the parenchymal cells using Perls’ technique, grade 0 representing absence of TABLE
I—MEAN(&sfgr;±S.E.M.)
VALUES IN CONTROLS
101 siderosis and grades 1-4 representing increasing degrees of iron deposition. 14 D.T.p.A.-chelatable body iron was determined using the chelation test previously described.7 Predicted total bodystorage iron (g.)= D.T.P.A.-chelatable iron (mg.) x 0-46, the standard deviation for such estimates being 112 g. in
patients with
iron
excess
whose
stores were
measured
by
venesection.7
Results
The findings in controls are shown in table i. Most of the specimens were obtained by post-mortem needle
TABLE II-FINDINGS IN PATIENTS WITH LIVER DISEASE AND VARIOUS IRON-LOADING DISORDERS
102 Additional quantitative measurecreased.10,11,15,1616 ments are desirable to confirm the presence of iron excess and to form a basis for rational management. Although acceptably precise estimates of body-iron stores are now possible with certain chelation tests, such measurements are essentially indirect, and the techniques, which entail radioisotope administration, are somewhat specialised. Liver biopsy is commonly done in patients thought to have iron excess, and the determination of iron concentration in such specimens, using the simple chemical procedure described here, has been found useful and should prove generally
practicable.
Relation between liver-iron concentration and D.T.P.A.-chelatable iron.
y=76-24x-35-98. (r=0963.) Predicted total body-storage iron (g.) D.T. P.A.-chelatable iron (mg.) x0.46.
=
sampling from patients who had died in hospital from myocardial infarction or cerebrovascular accident. None had a history of recent blood-loss, blood-transfusion, or surgical operation, and none had had liver disease. The average dry weight of the specimens was 9-4 mg. (range 41-193 mg.). In eight cases two specimens were examined, each from different parts of the liver; the standard deviation for a single specimen was 6-5 µg. iron per 100 mg. dry weight, which gave a coefficient of variation of 86°. The findings in patients with various forms of iron overload and in those with chronic liver disease are shown in table 11. The average dry weight of these specimens was 54 mg. (range 22-120 mg.). In 12 cases duplicate specimens were examined; the standard deviation for a single specimen was 123 µg. iron per 100 mg. dry weight (the mean iron concentration was 1738 µg. per 100 mg. dry weight), corresponding to a coefficient of variation of 7.1%. D.T.P.A.-chelatable iron was also determined in most of the cases listed in table 11, enabling total bodystorage iron to be predicted. As shown in the accompanying figure (in which logarithmic scales have been used for convenience) there was a highly significant correlation between liver-iron concentration and D.T.P.A.-chelatable iron (or predicted total body storage iron) (r=0.963; P "0001). The regression intercept did not differ significantly from the origin (P>0.6). The standard deviation for single estimates of total body-iron stores based on liver-iron concentration was 1 45 g. Discussion
Histochemical examination of liver-biopsy specimens is commonly used to assess the size of the iron stores and is valuable for screening purposes. The findings, however, are not always easy to interpret since appreciable amounts of stainable iron may be present when tissue-iron levels are normal or only slightly in-
The method measures total (as opposed to nonhaem) iron concentration, and the result therefore includes the hxmoglobin-iron content of the specimen. Assuming that liver tissue contains about 8% blood," the contribution due to hæmoglobin-iron may amount to 12 µg. per 100 mg. dry weight. This quantity is unimportant in the presence of iron excess but may be highly significant when there is storage iron depletion. Although we did not investigate liver biopsies from patients with chronic iron-deficiency anaemia, Weinfeld found that non-hæm iron in such patients was 2-10 µg. per 100 mg. dry weight, which would be expected to correspond to total iron concentrations of less than 25 µg. per 100 mg. dry weight. 11
The control observations accord well with pre-
viously published figures for normal human storage iron in adult liver obtained at necropsy.18 They also conform closely with values for non-haem iron in tissue obtained by excision biopsy 8,9and by aspiration needle-biopsy 10,11 from carefully selected controls undergoing surgery for uncomplicated biliary or peptic-ulcer disease. The failure to demonstrate a sex difference in our controls requires comment, however, since Weinfelda found that mean hepatic storage iron in postmenopausal women was significantly lower than in control men (though higher than in menstruating women). The explanation probably resides in the more advanced age of the females in our series (mean age 77, compared with mean age 60 in Weinfeld’s series). Close agreement between duplicate biopsies from the same liver has been previously demonstrated in controls,11 and it was reassuring to obtain similar concordance in specimens from patients with chronic liver disease, both with and without iron excess. The distribution of fat, fibrosis, and siderosis is often patchy, and greater sample variation might have been expected. However, the organ-distribution of storage iron may not be so uniform. Thus, in idiopathic hxmochromatosis deposition in hepatic parenchymal cells predominates and the spleen and bone-marrow are largely spared, whereas in hsemolytic states and transfusional overload the reticuloendothelial system bears the initial impact. Nevertheless, reticuloendothelial deposits are thought to be gradually redistributed to parenchymal stores in time, and the liver, which contains both components, has been regarded as generally most representative of total body iron content.19 Discrepancies between liver and bone-marrow do seem to be more striking at lower storage iron concentrations,and over a wide range of values a good correlation between the two sites was found." This is reflected here, in patients with various forms of iron
103
by the close correlation between liver iron concentration and D.T.p.A.-chelatable iron, for the latter has been shown to be highly correlated with total body storage iron as measured by venesection.7 Although our findings suggest that liver-iron concentration may be less precise than the chelation test for measuring total body-iron stores, the difference between the two methods would seem to be relatively excess,
FATE OF KIDNEY ALLOGRAFTS FROM CADAVERS WHOSE LIVERS WERE ALSO
TRANSPLANTED ALAN J. L. HART W. A. B. SMELLIE R. Y. CALNE
Department of Surgery, University of Cambridge
slight. A histogenetic relationship between iron excess and hepatic fibrosis has long been assumed in idiopathic and secondary hasmochromatosis, but experimental evidence in support of this concept has only lately been obtained.21 The quantitative aspects of the association have yet to be defined. Most patients with idiopathic hxmochromatosis seen at this hospital have iron loads of 10-15 g. (corresponding to liver-iron concentrations of 1-6-2-6% dry weight according to our data); fibrosis is usually heavy and nodules often present. This
Where both liver and kidneys are removed from a cadaver for transplantathe liver takes priority, so that there is a delay tion, before individual perfusion of the kidney can be started. However, experience with twenty renal allografts taken from 11 cadavers which were primarily liver donors suggests that kidneys from this source can be confidently used in renal transplant operations. Eighteen of these kidneys functioned immediately, and 7 weeks to 21 months after operation, fifteen of these are still functioning satisfactorily. All donors were
accords with the observation in Bantu siderosis that heavy fibrosis or cirrhosis is frequent when liver iron concentration exceeds 2% dry weight.22, 233 However, the actual amount of iron present may not be the only factor concerned in the pathogenesis of tissue injury, for only one of the four young patients with idiopathic haemochromatosis studied by Charlton et al.24 had appreciable fibrosis, though all had liver-iron contents exceeding 2%. Moreover, special factors seem to operate in pure transfusional overload (e.g., in aplastic anaemia), where fibrosis may be trivial despite iron excess of massive proportions (vide case 9). This has suggested that transfused hxmoglobin-iron may be relatively bland, perhaps because it is initially processed by the reticuloendothelial system, whereas excess iron accumulated via the gut is fibrogenic in much smaller amounts.25 Further understanding of the role of iron in the pathogenesis of liver damage must partly depend on correlating histological and chemical data in patients with iron-loading states, and in this respect valuable information may be obtained from prospective clinical studies with serial biopsies in patients with various forms of refractory anaemia.
respirator
We thank Dr. Peter Scheuer for the liver-biopsy interpretations, Dr. Michael Wills for the serum-iron estimations, and the many physicians who have referred patients to us.
Requests
for
reprints should be addressed to
S. S.
REFERENCES 1.
2. 3. 4.
Haskins, D., Stevens, A. R., Finch, S., Finch, C. A. J. clin. Invest. 1952, 31, 543. Pritchard, J. A., Mason, R. A. J. Am. med. Ass. 1964, 190, 897. Hynes, M. J. clin. Path. 1949, 2, 99. Balcerzak, S. P., Westerman, M. P., Heinle, E. W., Taylor, F. H.
Ann. intern. Med. 1968, 68, 518. Smith, P. M., Lestas, A. N., Miller, J. G. P., Dymock, I. W., Pitcher, C. S., Williams, R. Lancet, 1969, ii, 402. 6. Barry, M., Cartei, G. C., Sherlock, S. Gut, 1969, 10, 697. 7. Barry, M., Cartei, G. C., Sherlock, S. ibid. 1970, 11, 891. 8. Weinfeld, A. Acta med. scand. 1964, 177, suppl. 427. 9. Hallberg, L., Hedenberg, L., Weinfeld, A. Scand. J. Hœmat. 1966, 3, 85. 10. Weinfeld, A., Lundin, P., Lundvall, O. J. clin. Path. 1968, 21, 35. 11. Lundvall, O., Weinfeld, A., Lundin, P. Acta med. scand. 1969, 185, 5.
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Summary
cases.
Introduction
WE describe here the fate of twenty renal allografts taken from 11 cadavers which had also been used for liver transplantation. These kidneys were obtained under conditions which differ from those in cases where kidneys are the primary donor organ, since removal of the liver must take precedence because of its low tolerance to ischxmia. Methods In all cases the donors had irreversible brain damage, and ventilation was maintained artificially. Doctors independent of the transplant team decided in each case to stop mechanical respiration once irreversible brain death had been diagnosed. They informed the transplant team of this decision so that preparations could be made for a livergrafting operation. After mechanical respiration had been stopped, the surgeons waited until the circulation had ceased (as determined by doctors independent of the transplant team) before starting to remove the liver. Where the kidneys alone are to be transplanted, we remove organs as quickly as possible and perfuse them by the Gelin technique,l2with refrigerated Rheomacrodex until the renal-vein effluent is clear and following this with 200-300 ml. of a mixture of 10% fructose and 1-4% sodium bicarbonate. The kidneys are then packed in ice and placed in a thermos flask until the time of implantation in the recipient. However, where the liver is to be removed, the kidneys remain in situ while the liver is perfused via the portal vein with Hartmann’s solution, followed by a mixture of plasma, bicarbonate, and dextrose. The last two cases have also been perfused via the aorta in order that the hepatic artery may be perfused and also to acceler-
14.
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A. R.
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15. Finch, C. A., Bothwell, T. H. Archs intern. Med. 1961, 107, 807. 16. Morgan, E. H., Walters, M. N. J. clin. Path. 1963, 16, 101. 17. Scott, E. M., McCoy, R. H. Archs Biochem. 1944, 5, 349. 18. Powell, L. W. Australas. Ann. Med. 1966, 15, 110. 19. Bothwell, T. H., Finch, C. A. Iron Metabolism. London, 1962. 20. Gale, E., Torrance, J., Bothwell, T. H. J. clin. Invest. 1963, 42, 1076. 21. Lisboa, P. E. 5th Meeting of the European Association for the Study of the Liver, 1970; abstracts, p. 29. 22. Bothwell, T. H., Bradlow, B. A. Archs Path. 1960, 70, 279. 23. Bothwell, T. H., Isaacson, C. Br. med. J. 1962, i, 522. 24. Charlton, R. W., Abrahams, C., Bothwell, T. H. Archs Path. 1967, 83, 132. 25. Cappell, D. F., Hutchison, H. E., Jowett, M. J. Path. Bact. 1957, 74, 245.