BRITISH MEDICAL JOURNAL
611
3 SEPTEMBER 1977
14 Tanga, M R, et al, International Surgery, 1975, 60, 75. 15 Voogd, C E, et al, Mutation Research, 1974, 26, 483. 11; Rustia, M, and Shubik, P, Journal of the National Cancer Research Institute, 1972, 48, 721. 1 Berget, A, and Weber, T, Ugeskrift for Laeger, 1972, 40, 2085. I Ramsay, I D, British MedicalJournal, 1968, 4, 706. 9 Coxon, A, and Pallis, C A, J'ournal of Neurology, Neurosurgery and Psychiatry, 1976, 39, 403. 20 Long, C, editor, Biochemist's Handbook. London, Spon, 1961. 21 Placidi, G F, et al, Archives Internationales de Pharmacodynamie et de Therapie, 1970, 188, 168. 22 Ings, R M J, et al, Xenobiotica, 1975, 5, 223.
23 24
Rogulja, P V, et al, Acta Neuropathologica, 1973, 25, 36. Ings, R M J, et al, Biochemical Pharmacology, 1974, 23, 1421.
(Accepted 16 June 1977)
Departments of Neurology and Neurochemistry, General Hospital, and Department of Medicine, Royal Victoria Infirmary, Newcastle upon Tyne W G BRADLEY, DM, FRCP, professor of experimental neurology I J KARLSSON, MB, senior house officer in medicine C G RASSOL, PHD, research neurochemist
SHORT REPORTS Excessive alpha-fetoprotein concentrations in hepatocellular carcinoma We describe a patient with hepatocellular carcinoma, whose serum concentration of ac-fetoprotein exceeded 4 800 000 ,ug/l and produced an extra band on protein electrophoresis.
Case report A boy aged 16 was admitted to Frimley Park Hospital, Surrey, on 18 February 1976 complaining of severe right shoulder pain and abdominal distension of about two weeks' duration. He had a massively enlarged liver with considerable abdominal distension. Laboratory investigations gave the following results (normal ranges in parentheses): haemoglobin 12-1 g/dl (13-18 g/dl); total protein 76 g/l (60-74 g/l); albumin 43 g/l (36-47 g/l); total bilirubin 43 ,umol/l (2-5 mg/100 ml) (2-19 Ismol/l (0-12-1.1 mg/ 100 ml)); alkaline phosphatase 1866 U/I (80-300 U/1); aspartate aminotransferase 476 U!l (0-40 U/l); y-glutamyltransferase 792 U/l (0-45 U/1).
On 24 February laparoscopy showed blood-stained ascites and a grossly enlarged liver with multiple deposits in both lobes. Histological examination of a small biopsy specimen taken from a deposit confirmed the diagnosis of primary hepatocellular carcinoma. The serum concentration of a-fetoprotein (AFP) measured by radioimmunoassay was so abnormally high4 800 000 iLg/l (the normal range for healthy adults being < 25 ,Lg/l (< 25 ng/ ml))-that on serum protein electrophoresis on cellulose acetate it formed an extra band between albumin and ocl-globulin (see fig). The identification of the additional band was performed by the following techniques. A transfer technique1 2 in which the relevant band is cut out, placed on the surface of an agar plate, and left to diffuse against a specific antiserum applied to a strip of filter paper. The resulting precipitation arc may be visualised, stained, and localised. A modification of an immunofixation technique using cellulose acetate for separation3-after electrophoresis a specific antiserum (in this case anti-AFP) is applied to the separation pattern on agar gel and left to diffuse. Then the agar gel is washed, the "unfixed proteins" removed, and the gel stained. Both techniques confirmed that the additional band seen between the albumin and the xl fraction was oc-fetoprotein. Treatment with intravenous Aminosol and Intralipid was started on 27 February and continued until 6 March. The AFP concentration increased substantially during intravenous feeding, reaching a maximum value of 7 400 000 tsgll on 28 April. Despite treatment with x-irradiation to the liver area (300 rads), and subsequently a course of doxorubicin, he died on 7 July 1976.
(b) ,.e, 4
-g,ii*
tt.
~
~~1
A,AfJ;
|c
A
Anti AF-P, 4A t,' t._
Pbtient Al:bvrrr -
|
N tt 'AlIbumin~
19P
.li. . . ....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...........
y
Results of protein electrophoresis on cellulose acetate (a) showing presence of extra a-fetoprotein band (arrow) between albumin and ml band in patient's and neonate's serum, and absence of extra band in normal adult serum. Immunochemical confirmation of result (b) showing precipitation arc obtained with anti-a-fetoprotein (anti-AFP) serum using transfer technique (TR); electrophoretic pattern of patient's serum (EP); and precipitation band corresponding to additional fraction (X) obtained by immunofixation using anti-AFP serum (IF).
612
BRITISH MEDICAL JOURNAL
3 SEPTEMBER 1977
with osteomalacia and malabsorption, but no long-term studies have been reported. In our patient a good biochemical, histological, and symptomatic response was obtained after 14 months' treatment with oral 1 ocOHD3, despite the presence of only 30 cm of jejunum. This indicates that sufficient intestinal absorption of the analogue occurred to produce therapeutic blood levels and promote bone healing, even though a comparatively small dose was given. Little information is available on intestinal absorption of 1 xOHD3, although it seems likely that it is absorbed in a manner similar to that of the parent vitamin, and would thus be expected to be reduced in the presence of fat malabsorption.
Comment The maximum AFP concentration of 7 4 g/l recorded for our patient is, to our knowledge, the highest value ever reported. To appear as a separate band on routine electrophoresis, AFP must be nresent in a concentration exceeding 0-5 g/l. I
Kohn, J, Nature, 1957, 180, 986. Kohn, J, in Chromatographic and Electrophoretic Techniques, ed Ivor Smith, 4th edn, ch 5, p 120. London, Heinemann, 1900 and 1976. 3 Ritchie, R F, and Smith, R, Clinical Chemistry, 1976, 22 (4), 497. 2
(Accepted 14 April 1977)
Frimley Park Hospital, Frimley, Surrey GU16 SUJ A H AMERY, BSC, FRCS, consultant surgeon W C ALSTON, PHD, MRCPATH, consultant chemical pathologist
C
Vitamin D 120 000 units once weekly intramuscularly
11) E
Dihydrotachysterol 4 ml daily
la.OHD3
1,ug 2jug
Department of Chemical Pathology, Queen Mary's Hospital, Roehampton, London SW1 55PN J KOHN, FRCPATH, DCP, director, protein reference laboratory
Department of Nuclear Medicine, St Luke's Hospital, Guildford, Surrey W F WHITE, MB, FRCR, consultant radiotherapist
1
1_ 2 %A
80O
.
0_
Treatment of osteomalacia with oral 1-alpha-hydroxyvitamin D3 in a patient with malabsorption Vitamin D is now known to undergo 25-hydroxylation in the liver and subsequently 1 a-hydroxylation in the kidney to form 1,25dihydroxyvitamin D, (1,25(OH)2D), before exerting its biological effects.' This knowledge has stimulated investigation into possible therapeutic applications of vitamin D metabolites and their synthetic analogues in metabolic bone disease. 1 a-hydroxyvitamin D3 (1 ocOHD3), an analogue of 1,25(OH)2D, has been used to treat renal osteodystrophy2 but there are no descriptions of its long-term use in osteomalacia due to malabsorption. This report describes the response to small oral doses of 1 aOHD3 in a patient with severe osteomalacia after massive small intestinal resection.
Case history, methods, and results The patient, a housewife now aged 33 years, underwent resection of all the small intestine excepting 30 centimetres of jejunum after a volvulus in 1971. Osteomalacia was diagnosed in January 1973, and details of treatment are shown in the figure. Renal and hepatic function were normal. Transiliac bone biopsies were performed in January 1973, March 1975, November 1975, and May 1976. Eight,um undecalcified sections were assessed quantitatively using both a Quantimet 720 automated image analyser and an eye-piece graticule. Calcification fronts were demonstrated by tetracycline fluorescence on 10 ,um unstained sections following oral administration of demeclocycline (Ledermycin) 48 hours before the biopsy. Serum calcium, phosphate, and alkaline phosphatase concentrations were measured on an SMA 12/60
Autoanalyser. Biochemical and histological data are shown in the figure. Serum calcium and phosphate concentrations were initially normal and remained so throughout the treatment period. The raised alkaline phosphatase returned to normal for the first time seven months after starting locOHD3. After two years of parenteral vitamin D2 treatment calcification fronts had become normal and osteoid volume had decreased, although still abnormally raised. After six months of treatment with l1xOHD3 osteoid volume had become normal. Histological and biochemical improvement during lcxOHD3 treatment was accompanied by disappearance of bone pain which had been present for three years.
Discussion Biochemical and histological improvement after short-term oral3 and intravenous4 treatment with 1 cxOHD3 has been shown in patients
Details of treatment and response of serum alkaline phosphatase concentration and bone histology. Dashed horizontal line indicates upper limit of normal for serum alkaline phosphatase.
The therapeutic use of this analogue has several theoretical and practical advantages over parenteral vitamin D treatment. Firstly, oral treatment is more acceptable than parenteral treatment to most patients. Secondly, even though some histological improvement was seen after almost two years of parenteral vitamin D treatment, a comparable further improvement was seen after only six months of laOHD, treatment, suggesting a greater therapeutic effecitiveness of 1 ocOHD3 compared with the parent vitamin, even though at the doses used the weight equivalent of the latter was 215 times greater. Thirdly, although hypercalcaemia may complicate either form of treatment, it can be much more rapidly reversed after 1 ocOHD3 treatment.5 Hypercalcaemia has not occurred in our patient, and possibly malabsorption may protect against this complication. We conclude that 1 ocOHD3, given orally, may prove to be of value in treating osteomalacia in patients with malabsorption. We thank Adrian Webb for technical help. JEC gratefully acknowledges the support of the Endowments Fund, St Thomas's Hospital. We thank Leo Laboratories Ltd, for supplies of synthetic lIcOHD3. I 2
Hoiick, M F, et al, Biochemistry, 1971, 10, 2799.
Coburn, J W, Hartenbower, D L, and Brickman, A S, American Journal of
Clinical Nutrition, 1976, 29, 1283. 3Bordier, P J, et al, in Vitamin D and Problems Related to Uraemic Bone Disease, ed AW Norman, et al, p 133, Berlin, W de Gruyter, 1975. 4Bordier, P J, et al, New England Journal of Medicine, 1974, 291, 866. 5 Kanis, J A, and Russell, R G G, British Medical3Journal, 1977, 1, 78.
(Accepted 14 April 1977) Gastrointestinal Laboratory, Rayne Institute, and Department of Surgical Pathology, St Thomas's Hospital, London SE1 JULIET E COMPSTON, BSC, MRCP, senior research registrar L W L HORTON, BM, MRCP, lecturer in pathology J R TIGHE, MD, FRCPATH, professor of histopathology
611
3 SEPTEMBER 1977
14 Tanga, M R, et al, International Surgery, 1975, 60, 75. 15 Voogd, C E, et al, Mutation Research, 1974, 26, 483. 11; Rustia, M, and Shubik, P, Journal of the National Cancer Research Institute, 1972, 48, 721. 1 Berget, A, and Weber, T, Ugeskrift for Laeger, 1972, 40, 2085. I Ramsay, I D, British MedicalJournal, 1968, 4, 706. 9 Coxon, A, and Pallis, C A, J'ournal of Neurology, Neurosurgery and Psychiatry, 1976, 39, 403. 20 Long, C, editor, Biochemist's Handbook. London, Spon, 1961. 21 Placidi, G F, et al, Archives Internationales de Pharmacodynamie et de Therapie, 1970, 188, 168. 22 Ings, R M J, et al, Xenobiotica, 1975, 5, 223.
23 24
Rogulja, P V, et al, Acta Neuropathologica, 1973, 25, 36. Ings, R M J, et al, Biochemical Pharmacology, 1974, 23, 1421.
(Accepted 16 June 1977)
Departments of Neurology and Neurochemistry, General Hospital, and Department of Medicine, Royal Victoria Infirmary, Newcastle upon Tyne W G BRADLEY, DM, FRCP, professor of experimental neurology I J KARLSSON, MB, senior house officer in medicine C G RASSOL, PHD, research neurochemist
SHORT REPORTS Excessive alpha-fetoprotein concentrations in hepatocellular carcinoma We describe a patient with hepatocellular carcinoma, whose serum concentration of ac-fetoprotein exceeded 4 800 000 ,ug/l and produced an extra band on protein electrophoresis.
Case report A boy aged 16 was admitted to Frimley Park Hospital, Surrey, on 18 February 1976 complaining of severe right shoulder pain and abdominal distension of about two weeks' duration. He had a massively enlarged liver with considerable abdominal distension. Laboratory investigations gave the following results (normal ranges in parentheses): haemoglobin 12-1 g/dl (13-18 g/dl); total protein 76 g/l (60-74 g/l); albumin 43 g/l (36-47 g/l); total bilirubin 43 ,umol/l (2-5 mg/100 ml) (2-19 Ismol/l (0-12-1.1 mg/ 100 ml)); alkaline phosphatase 1866 U/I (80-300 U/1); aspartate aminotransferase 476 U!l (0-40 U/l); y-glutamyltransferase 792 U/l (0-45 U/1).
On 24 February laparoscopy showed blood-stained ascites and a grossly enlarged liver with multiple deposits in both lobes. Histological examination of a small biopsy specimen taken from a deposit confirmed the diagnosis of primary hepatocellular carcinoma. The serum concentration of a-fetoprotein (AFP) measured by radioimmunoassay was so abnormally high4 800 000 iLg/l (the normal range for healthy adults being < 25 ,Lg/l (< 25 ng/ ml))-that on serum protein electrophoresis on cellulose acetate it formed an extra band between albumin and ocl-globulin (see fig). The identification of the additional band was performed by the following techniques. A transfer technique1 2 in which the relevant band is cut out, placed on the surface of an agar plate, and left to diffuse against a specific antiserum applied to a strip of filter paper. The resulting precipitation arc may be visualised, stained, and localised. A modification of an immunofixation technique using cellulose acetate for separation3-after electrophoresis a specific antiserum (in this case anti-AFP) is applied to the separation pattern on agar gel and left to diffuse. Then the agar gel is washed, the "unfixed proteins" removed, and the gel stained. Both techniques confirmed that the additional band seen between the albumin and the xl fraction was oc-fetoprotein. Treatment with intravenous Aminosol and Intralipid was started on 27 February and continued until 6 March. The AFP concentration increased substantially during intravenous feeding, reaching a maximum value of 7 400 000 tsgll on 28 April. Despite treatment with x-irradiation to the liver area (300 rads), and subsequently a course of doxorubicin, he died on 7 July 1976.
(b) ,.e, 4
-g,ii*
tt.
~
~~1
A,AfJ;
|c
A
Anti AF-P, 4A t,' t._
Pbtient Al:bvrrr -
|
N tt 'AlIbumin~
19P
.li. . . ....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...........
y
Results of protein electrophoresis on cellulose acetate (a) showing presence of extra a-fetoprotein band (arrow) between albumin and ml band in patient's and neonate's serum, and absence of extra band in normal adult serum. Immunochemical confirmation of result (b) showing precipitation arc obtained with anti-a-fetoprotein (anti-AFP) serum using transfer technique (TR); electrophoretic pattern of patient's serum (EP); and precipitation band corresponding to additional fraction (X) obtained by immunofixation using anti-AFP serum (IF).
612
BRITISH MEDICAL JOURNAL
3 SEPTEMBER 1977
with osteomalacia and malabsorption, but no long-term studies have been reported. In our patient a good biochemical, histological, and symptomatic response was obtained after 14 months' treatment with oral 1 ocOHD3, despite the presence of only 30 cm of jejunum. This indicates that sufficient intestinal absorption of the analogue occurred to produce therapeutic blood levels and promote bone healing, even though a comparatively small dose was given. Little information is available on intestinal absorption of 1 xOHD3, although it seems likely that it is absorbed in a manner similar to that of the parent vitamin, and would thus be expected to be reduced in the presence of fat malabsorption.
Comment The maximum AFP concentration of 7 4 g/l recorded for our patient is, to our knowledge, the highest value ever reported. To appear as a separate band on routine electrophoresis, AFP must be nresent in a concentration exceeding 0-5 g/l. I
Kohn, J, Nature, 1957, 180, 986. Kohn, J, in Chromatographic and Electrophoretic Techniques, ed Ivor Smith, 4th edn, ch 5, p 120. London, Heinemann, 1900 and 1976. 3 Ritchie, R F, and Smith, R, Clinical Chemistry, 1976, 22 (4), 497. 2
(Accepted 14 April 1977)
Frimley Park Hospital, Frimley, Surrey GU16 SUJ A H AMERY, BSC, FRCS, consultant surgeon W C ALSTON, PHD, MRCPATH, consultant chemical pathologist
C
Vitamin D 120 000 units once weekly intramuscularly
11) E
Dihydrotachysterol 4 ml daily
la.OHD3
1,ug 2jug
Department of Chemical Pathology, Queen Mary's Hospital, Roehampton, London SW1 55PN J KOHN, FRCPATH, DCP, director, protein reference laboratory
Department of Nuclear Medicine, St Luke's Hospital, Guildford, Surrey W F WHITE, MB, FRCR, consultant radiotherapist
1
1_ 2 %A
80O
.
0_
Treatment of osteomalacia with oral 1-alpha-hydroxyvitamin D3 in a patient with malabsorption Vitamin D is now known to undergo 25-hydroxylation in the liver and subsequently 1 a-hydroxylation in the kidney to form 1,25dihydroxyvitamin D, (1,25(OH)2D), before exerting its biological effects.' This knowledge has stimulated investigation into possible therapeutic applications of vitamin D metabolites and their synthetic analogues in metabolic bone disease. 1 a-hydroxyvitamin D3 (1 ocOHD3), an analogue of 1,25(OH)2D, has been used to treat renal osteodystrophy2 but there are no descriptions of its long-term use in osteomalacia due to malabsorption. This report describes the response to small oral doses of 1 aOHD3 in a patient with severe osteomalacia after massive small intestinal resection.
Case history, methods, and results The patient, a housewife now aged 33 years, underwent resection of all the small intestine excepting 30 centimetres of jejunum after a volvulus in 1971. Osteomalacia was diagnosed in January 1973, and details of treatment are shown in the figure. Renal and hepatic function were normal. Transiliac bone biopsies were performed in January 1973, March 1975, November 1975, and May 1976. Eight,um undecalcified sections were assessed quantitatively using both a Quantimet 720 automated image analyser and an eye-piece graticule. Calcification fronts were demonstrated by tetracycline fluorescence on 10 ,um unstained sections following oral administration of demeclocycline (Ledermycin) 48 hours before the biopsy. Serum calcium, phosphate, and alkaline phosphatase concentrations were measured on an SMA 12/60
Autoanalyser. Biochemical and histological data are shown in the figure. Serum calcium and phosphate concentrations were initially normal and remained so throughout the treatment period. The raised alkaline phosphatase returned to normal for the first time seven months after starting locOHD3. After two years of parenteral vitamin D2 treatment calcification fronts had become normal and osteoid volume had decreased, although still abnormally raised. After six months of treatment with l1xOHD3 osteoid volume had become normal. Histological and biochemical improvement during lcxOHD3 treatment was accompanied by disappearance of bone pain which had been present for three years.
Discussion Biochemical and histological improvement after short-term oral3 and intravenous4 treatment with 1 cxOHD3 has been shown in patients
Details of treatment and response of serum alkaline phosphatase concentration and bone histology. Dashed horizontal line indicates upper limit of normal for serum alkaline phosphatase.
The therapeutic use of this analogue has several theoretical and practical advantages over parenteral vitamin D treatment. Firstly, oral treatment is more acceptable than parenteral treatment to most patients. Secondly, even though some histological improvement was seen after almost two years of parenteral vitamin D treatment, a comparable further improvement was seen after only six months of laOHD, treatment, suggesting a greater therapeutic effecitiveness of 1 ocOHD3 compared with the parent vitamin, even though at the doses used the weight equivalent of the latter was 215 times greater. Thirdly, although hypercalcaemia may complicate either form of treatment, it can be much more rapidly reversed after 1 ocOHD3 treatment.5 Hypercalcaemia has not occurred in our patient, and possibly malabsorption may protect against this complication. We conclude that 1 ocOHD3, given orally, may prove to be of value in treating osteomalacia in patients with malabsorption. We thank Adrian Webb for technical help. JEC gratefully acknowledges the support of the Endowments Fund, St Thomas's Hospital. We thank Leo Laboratories Ltd, for supplies of synthetic lIcOHD3. I 2
Hoiick, M F, et al, Biochemistry, 1971, 10, 2799.
Coburn, J W, Hartenbower, D L, and Brickman, A S, American Journal of
Clinical Nutrition, 1976, 29, 1283. 3Bordier, P J, et al, in Vitamin D and Problems Related to Uraemic Bone Disease, ed AW Norman, et al, p 133, Berlin, W de Gruyter, 1975. 4Bordier, P J, et al, New England Journal of Medicine, 1974, 291, 866. 5 Kanis, J A, and Russell, R G G, British Medical3Journal, 1977, 1, 78.
(Accepted 14 April 1977) Gastrointestinal Laboratory, Rayne Institute, and Department of Surgical Pathology, St Thomas's Hospital, London SE1 JULIET E COMPSTON, BSC, MRCP, senior research registrar L W L HORTON, BM, MRCP, lecturer in pathology J R TIGHE, MD, FRCPATH, professor of histopathology
