391
original pepsin-trypsin digest. That the lymphocyte reactivity is restricted to cceliac patients suggests immunological sensitisation. This may result from an inin the
herent
Coeliac
General medical
patients
Gastroenterology patients
patients Fig, +-Stimulation index
of
lymphocytes
cultured for 48 h with P.H.A.
or
acquired difference in the immune
response to
antigenic determinants of gluten. Alternatively it may result from the stimulation of immunocompetent cells in the jejunal mucosa by antigens that remain after the incomplete degradation of gluten by cœliac mucosa. The ability of a2 to cause an enteropathy in patients suggests that lymphocyte sensitisation may have a role in the pathogenesis of the disease. Indeed, it is known that T lymphocytes have a role in the production of other forms of villous atrophy. Thus studies on animal models, including mouse intestinal allograft rejection," and the colonization of mouse jejunum by the nematode Nippostrongylus braziliensis12 show that villous atrophy, similar to that found in coeliac patients, does not occur in mice deprived of T lymphocytes by thymectomy. Further work is necessary to determine the usefulness of the assay as a screening test for coeliac disease and to attempt to purify further the active component ofB2. We thank Dr W. Jacobson for suggesting these experiments; Dr Max Perutz for initiating our collaboration; Prof. E. Lennox and Dr T. Pearson for helpful advice; and Dr A. P. Dick and Dr J. Hunter for allowing us to study their patients. We thank the M.R.C. for financial help including a Trainee Fellowship for K.S. Requests for reprints should be addressed to K. S., M.R.C. Laboratory of Molecular Biology, University Postgraduate Medical School, Hills Road, Cambridge CB2 2QH. REFERENCES
zer’s fraction
fraction
reproducibly stimulated D,S.A. synthesis in lymphocytes from coeliac patients. However, a subfraction, B2, prepared by sephadex G-50 gel filtration consistently stimulated while Bland B3 did not do so. A dose-response titration using this B2 subfraction is shown in fig. 1. Maximum stimulation was ill nor
B
obtained with 10 µg/ml final concentration of this material. A time-course study (using 10 µg/ml B2) showed peak stimulation at 48 h (fig. 2). Lymphocytes from 4 cœliac patients) which responded well to B2 did not respond to other dietary proteins including &agr;-lactalbumin, &bgr;-lactoglobulin, and casein (data not shown). The response of lymphocytes from a series of cceliac patients and controls to B2 was then measured and is shown in fig. 3. Lymphocyte stimulation by P.H.A. (a potent T-cell mitogen) was measured concurrently (fig. 4. There is a clear difference in the reactivity of the lymphocytes from coeliac patients to B2 when compared with ooth general medical and gastroenterological controls P 001 in both cases). There is no significant difference between any of these groups in the degree of lymphocyte rssponsueness to P.H.A. at 48 h. The various subfractions are at present being tested for their damaging properties by feeding experiments with cceliac volunteers followed by peroral jejunal nopsy, Preliminary results show that B2 and B3 cause istological abnormalities in jejunal mucosa whilst Bl1
joes not.9 Discussion
.
It is clear that lymphocytes from coeliac patients are ’.imutated by the B2 subfraction of gluten whereas lymphocytes from the control groups are not. It may be that ,. preparation of the B2 subfraction concentrates the stimulatory component or removes an inhibitor present
1. 2. 3.
Shiner, M. Gut, 1973, 14, 1. Marsh, M. N. ibid. 1975, 16, 665. Keuning, J. J., Pena, J., Van Leeuwen, A., Van Hoof, J. P., Van Rood, J. J. Lancet, 1976, i, 506. 4. Holmes, G. K. T., Asquith, P., Cooke, W. T. Gut, 1972, 13, 324. 5. Holmes, G. K. T., Asquith, P., Cooke, W. T. Clin. exp. Immun. 1976, 24, 259. 6. Morganroth, J., Watson, D., French, A. B. J. dig. Dis. 1972, 17, 205. 7. Frazer, A. C., Fletcher, R. F., Ross, A., Shaw, B., Sammons, H. G., Schneider, R. Lancet, 1959, u, 252. 8. Dissayanake, A. S., Jerrome, D. W., Offord, R. E., Truelove, S. C., Whitehead, R. Gut, 1974, 15, 931. 9. Anand, B. S., Piris, J., Offord, R. E., Truelove, S. C. Unpublished. 10. Hartzmann, R. H. Cell. Immun. 1972, 4, 127. 11. Macdonald, T. T., Ferguson, A. Gut, 1976, 17, 81. 12. Ferguson, A., Jarrett, E. E. E. ibid. 1975, 16, 114.
COMPARISON OF VITAMIN D AND 25-HYDROXY-VITAMIN-D IN THE THERAPY OF PRIMARY BILIARY CIRRHOSIS* B. WAGONFELD MERRY BOLT JAMES L. BOYER
JAMES
Section
BERNARD A. NEMCHAUSKY JEAN VANDER HORST IRWIN H. ROSENBERG
of Gastroenterology and Liver Study Unit, University of Chicago, Chicago, Illinois 60637, U.S.A.
Skeletal demineralisation and low serum concentrations of 25-hydroxy-vitamin-D were observed in patients with primary biliary cirrhosis. Neither oral nor parenteral vitamin-D increased
Summary
25-hydroxy-vitamin-D
in
serum
or
prevented further 25-hydroxy-
skeletal demineralisation. In contrast, oral
was presented in part at the annual meeting of the AmeriAssociation for the Study of Liver Disease, November, 1975.
*This paper can
392 vitamin-D increased serum-25-hydroxy-vitamin D concentrations in all patients, and bone mineral content either improved or stabilised in all but one. 25-hydroxyvitamin-D may be the preferred form of vitamin-D therapy in primary biliary cirrhosis. Introduction To function physiologically vitamin D must be hydroxylated in the liver to 25-hydroxy-vitamin-D’ and sub-
sequently by the kidney to lx, 25-dihydroxy-vitamin-D.2 Therefore defective vitamin-D utilisation and bone disease might be expected as a complication of either chronic hepatic or chronic renal disease. Bone disease associated with renal disease can be corrected by therapy with 1&agr;, 25-dihydroxy-vitamin-D.34 While osteoporosis and osteomalacia occur in association with chronic hepatic disease, the metabolic basis for this complication has not been clarified.56 Primary biliary cirrhosis, a disease of unknown aetiology characterised by severe derangement of hepatic excretory function and progressive cirrhosis, is commonly complicated by skeletal demineralisation which often progresses despite therapy with high doses of oral or parenteral vitamin D. 7-12 Malabsorption of vitamin D has been found in primary biliary cirrhosis.13 However, the failure of parenteral vitamin D to regularly improve either skeletal demineralisation or calcium malabsorption7-12 suggests that a defect in hepatic metabolism of vitamin D rather than intestinal malabsorption may be the major cause of the skeletal complications of primary biliary cirrhosis. The recently synthesised hepatic metabolite of vitamin D, 25-hydroxy-vitamin-D14 might circumvent such a defect in hepatic metabolism. We therefore determined serum-25-hydroxy-vitamin-D and bone mineral content in primary biliary cirrhosis and compared the therapeutic effect of oral and parenteral vitamin D with that of 25-hydroxy-vitamin-D.
in the within 2%."
ment
same
individual
on
repeated determinationsr
Serum-25-hydroxy-vitamin-D levels were determined by th, competitive protein-binding method of Haddad and Chyu’ with the following modifications. Kidney cytosol binder was prepared from non-rachitic rats. The specific activity of the labelled 25-hydroxy-vitamin-D was 29 Ci/mmole. After sili, cic-acid chromatography, samples were dissolved in ethanol so that aliquots of 50 and 100 µl could be taken for the final binding step. This modification reduced the error mtroduced b evaporation. Improved agreement between duplicates was obtained when the samples were kept in ice during binding and after the addition of dextran-coated charcoal. Samples froma serum pool were reassayed during each run and a mean difference of ±8.1% was obtained. When nine duplicate serumsamples were analysed on different days the mean difference of paired duplicates was ±98%. In our laboratory the mean serum concentration of 25-hydroxy-vitamin-D in seventeen normal volunteers
was
2 3.8 + 5.64 ng/ml.
Patients and Methods
Eight female patients aged 35-62 years were included in study. The patients had had symptoms of primary biliary cirrhosis for 15-125 months at the start of therapy. Seven patients had positive tests for antimitochondrial antibody. Five were clinically jaundiced and had steatorrhcea greater than lOg a day. Only three had advanced cirrhosis morphologically. this
All but one had entered the menopause at least 5 years before the onset of the study. Two patients had severe bone pain. Skeletal mineralisation was assessed in each patient by two criteria: (1) metacarpal cortical thickness, (2) proton-beam absorption. Fine-detail radiographs of the hand were obtained using a fine-grain industrial film (Kodak type M) and were reviewed with optical magnification at 4-10 times by the method of Genant.15 The combined cortical thickness of the left second metacarpal was determined using the technique of Garn.16 Bone mineral content was determined by the technique developed and standardised by Lanzl and Strandjord.17 This method consists of measuring the transmission of a narrow colimated beam of radiation which emanates from an iodine-125 source and is passed through the diaphysis of the middle phalynx of the third digit of the left hand. The transmitted beam is detected by a sodium-iodine scintillation crystal connected to a photo multiplier, discriminator, and scaler. The result is expressed as the linear absorption coefficient,bone, cm-i, after adjustment for bone width and correction for soft tissue and is a measure of the mineral content of both cortical and medullary phalangeal bone. Normal values for our institution have previously been determined from a study of three hundred controls. The reproducibility of the measure-
Summary of the
response to treatment in all
patients.
panel A, therapy with oral vitamin-D is shown in solid lines ai. therapy with parenteral vitamin-D in broken lines. Only those patient In
who had pre and post treatment values are included. Panel B shi,1 the change in linear absorption coefficient of bone expressed as a K cent of age and sex matched control values during vitamin-D therap Panel c shows the response in serum-25-hydroxy-vitamin-D follo therapy with oral 25-hydroxy-vitamin-D. Panel D shows the change’ linear absorption coefficient of bone, expressed as a percent of age sex matched control values after 3-6 months therapy with
25-hydroxy-vitanun-D.
393 25-HYDROXY-VITAMIN-D AND SKELETAL MINERALISATION (MEAN i S.D.) IN PRIMARY BILIARY CIRRHOSIS
CIRCULATING
signs, symptoms, or laboratory data consistent with hypervitaminosis D, doses were subsequently adjusted to maintain circulating vitamin D levels near the control range. On this regimen the linear absorption coefficient of bone either increased
or
remained stable in all but
one
patient (panel D). Discussion Control values for 25-hydroxy-vitamin-D were obtained from normal volunteers ’n late wmter. All 6 patients were studied before any vitamm-D therapy. ’age, sex, and race matched control values for metacarpal cortical thickness 16 were obtained from the data of Garn. sex matched for linear absorption coefficient were control values Ageand 17 obtamedfrom the data of Lanzl and Strandjord. Bumbers in parentheses are the number of patients studied.
Results
Results of the
measurement
of
metacarpal
cortical
thickness, linear absorption coefficients, and serum-25-
hydroxy-vitamin-D for the patients with primary biliary cirrhosis are shown in the accompanying table and are contrasted with the values for the healthy age and sex matched controls. The patients had a mean cortical thickness and mean absorption coefficient significantly less than those of the age and sex matched controls (Student’s t test). In six untreated patients with primary biliary cirrhosis the mean serum-25-hydroxy-vitamin-D level was 4.4±3.4 ng/ml, significantly less than the normal volunteers (23.815.6 ng/ml) (Student’s t test). All patients’ values were below the lowest normal value and two patients had levels unmeasurable by our technique (< 2
neml). At the onset of the study linear absorption coefficient of bone was normal for age and sex. During the 3 months of therapy with parenteral vitamin D, the linear absorption coefficient of bone decreased 10%, a decrease far greater than would be expected by assay variability or the normal aging process. After oral 25-hydroxy-vitamin-D therapy for 3 months, the linear absorption coefficient increased by 5%, a change greater than the variability of the measurement. The response to treatment is summarised in the accompanying figure. Seven patients were treated for 3-6 months with either daily oral vitamin D in doses from 1000 to 50 000 units or monthly parenteral vitamin D 100 000 to 500 000 units subcutaneously. As shown in panel A, none of the six patients in whom both pre and post treatment samples were available responded to either oral or parenteral vitamin D with an elevation in the serum level of 25-hydroxy-vitamin-D to control range. One patient not shown in panel A because her pretreatment concentration was not available did have a normal serum-25-hydroxy-vitamin-D after parenteral vitamin-D therapy. At the end of the 3-6 month treatment period, the linear absorption coefficient of bone was reassessed in each patient (panel B) and a decrease in bone mineral content greater than 2% occurred in all but 2 patients. One patient showed no change. Only the patient with a normal serum-25-hydroxv-vitamin-D after vitamin-D therapy showed improvement in bone mineral content. In contrast to conventional vitamin D, all patients responded to daily oral therapy of 100-200 µg of 25-25-hydroxy-vitamin-D with a rise in serum-25-hydroxy-vitamin-D to normal or high values (panel c). Although no patient demonstrated
Previous studies of the skeletal complications of biliary cirrhosis have described the occurrence of osteomalacia and osteoporosis,19 intestinal calcium malabsorption,2o and low serum-25-hydroxy-vitamin-D.21Despite well-documented malabsorption of vitamin D in primary biliary cirrhosis13 Ajdukiewicz et al.12 have emphasised that symptomatic bone pain and osteomalacia may progress despite long-term therapy with parenteral vitamin D and have suggested that factors other than malabsorption such as decreased hepatic 25-hydroxylation may be of importance.21 Our studies record low serum-25-hydroxy-vitamin-D in patients with primary biliary cirrhosis and show that either oral or parenteral vitamin D in high doses usually fails to correct this abnormality. Since skeletal demineralisation progressed despite parenteral vitamin-D therapy, but stabilised or improved on oral therapy with 25-hydroxy-vitamin-D, impaired hepatic hydroxylation of vitamin D rather than malabsorption would seem to be the major factor in the failure of conventional vitamin-D therapy in primary biliary cirrhosis. Therapy with pharmacological doses of 25-hydroxy-vitamin-D might be expected to cause much greater rises in serum-25-hydroxy-vitamin-D if malabsorption was not present. However, the inability of doses of vitamin D as high as 12.5mg given parenterally to raise serum-25-hydroxy-vitamin-D in vitamin D deficient patients conclusively demonstrates that failure of hydroxylation is a major cause of low 25-hydroxy-vitamin-D levels in these patients and may be an important factor in the vitamin D unresponsiveness in liver disease. Theoretically, liver disease could effect not only the formation, but also hepatic release of 25-hydroxy-vitamin-D. Also, the patient with liver disease might additionally have a defect in renal hydroxylation or may even have some degree of end organ unresponsiveness. The present study does not exclude these other possibilities. These studies show that oral therapy with 25-hydroxy-vitamin-D may circumvent an abnormality in hepatic metabolism of vitamin D in primary biliary cirrhosis and might prevent or ameliorate the hepatic osteodystrophy which often complicates its course. Treatment with 25-hydroxy-vitamin-D should proceed with care until long-term studies determine whether histologic improvement of skeletal demineralisation is also achieved. We thank Dr
roxy-vitamin-D
John Hinman of the Upjohn Company for the 25-hydused in this study and Ms Kathy Linneman for secre-
tarial assistance. This work was supported in part by U.S.P.H.S. Training Grant AM-05147, General Clinical Research Grant RR-55, and N.I.H. Fellowships AM-05150 (Dr Wagonfeld) and AM-05248 (Dr Nemchausky), and by the Gastrointestinal Research Foundation. Dr Boyer is the recipient of Academic Career Development Award AM-70218 from the Institute of Arthritis, Metabolism, and Digestive Diseases. The bone-density apparatus is maintained under Contract E(11-1)-69 from the United States Energy Research and Development Administration.
References overleaf.
394 EFFECT OF PECTIN ON SERUM LIPIDS AND
LIPOPROTEINS, WHOLE-GUT TRANSIT-TIME, AND STOOL WEIGHT P. N. DURRINGTON C. H. BOLTON
A. P. MANNING M. HARTOG
University of Bristol Department of Medicine, Bristol Royal
Infirmary, Bristol BS2 8HW Pectin (12g daily with meals) was taken by twelve healthy men aged 22-45 (mean 25yr) for 3 weeks. This produced a statistically significant mean decrease in total serum-cholesterol of 0.48±0.18 mmol/l (±S.E.M.)—i.e., 7.9±2.6%. The decrease was largely due to a reduction in serum-lowdensity-lipoprotein-cholesterol of 0.45±0.19 mmol/l accompanied by a fall in serum-apolipoprotein-B of 0.11±0.04 g/l. During pectin administration wet stool weight increased from 150±10 to 186±15 g/24 h. There was no clear change in total serum-triglycerides, serum very low density lipoproteins, or in whole-gut transit-
Summary
time.
Introduction PECTIN is the partially methoxylated polymer of galacturonic acid present in the white inner rind and connective tissue of apples, citrus, and various other fruits. Pectin reduced total serum-cholesterol in healthy individuals when taken orally.1-3 However, the effects of pectin on serum triglycerides and lipoproteins and on intestinal function have not previously been reported.
Methods
Subjects and Design of Study Twelve non-obese, healthy male volunteers aged 22-45 (mean 25yr) gave their informed consent to the study, which had been approved by an ethical committee. All the men maintained their normal diet throughout the investigation and
Ponchon, G., Kennan, A. L., DeLuca, H. F. J. clin. Invest. 1969, 48, 2032. Gray, R., Boyle, I., DeLuca, H. F. Science, 1971, 172, 1232. Brickman, A. S., Coburn, J. W., Norman, A. W. New Engl. J. Med. 1972, 287, 891. 4. Brickman, A. S., Coburn, J. W., Massry, S. G., Norman, A. W. Ann. intern. Med. 1974, 80, 161. 5. Collession, L., Grilliat, J. P., Mathieu, J. Presse méd. 1965, 73, 2571. 6. Meunier, P., Dechavanne, M., Prost, G. Lyon Méd. 1973, 229, 1089. 7. Kehayoglou, A. K., Holdsworth, C. D., Agnew, J. E., Whelton, M. J., Sherlock, S. Lancet, 1968, i, 715. 8. Ahrens, E. H., Jr., Payne, M. A., Kunkel, H. G., Eisenmenger, W. J., Blondheim, S. H. Medicine, 1950, 29, 299. 9. Sherlock, S. Gastroenterology, 1959, 37, 574. 10. Wessler, S., Avioli, L. A. J. Am. med. Ass. 1968, 206, 1285. 11. Avioli, L. A., Haddad, J. G. Metobolism, 1973, 22, 507. 12. Ajdukiewicz, A. B., Agnew, J. E., Byers, P. D., Wills, M. R., Sherlock, S. Gut, 1974, 15, 788. 13. Thompson, G. W., Lewis, B., Booth, C. C. J. clin. Invest. 1966, 45, 94. 14. Blunt, J. W., DeLuca, H. F. Biochemistry, 1969, 8, 671. 15. Genant, H. K., Doi, K., Mall, J. C. Invest. Radiol. 1975, 10, 160. 16. Garn, S. The Earlier Gain and the Later Loss of Cortical Bone in Nutritional Perspective; Springfield, Illinois, 1970. 17. Lanzl, L. H., Strandjord, N. Symposium on Low-energy and Gamma Ray Sources and Application, Chicago, Illinois, October, 1964. 18. Haddad, J. G., Chyu, K. J. J. clin. Endocr. 1971, 33, 992. 19. Atkinson, M., Nordm, B. E. C., Sherlock, S. Q. Jl Med. 1956, 25, 299. 20. Whelton, M. J., Kehayoglou, A. K., Agnew, J. E., Turnberg, L. A., Sherlock, S. Gut, 1971, 12, 978. 21. Ajdukiewicz, A. J., Hamlyn, A. N., Preece, M. A., Ribot, C. A., O’Riordan, J. L. H., Sherlock, S. Digestion, 1974, 10, 332. 1. 2. 3.
avoided excessive alcohol intake. No medication was taken for at least 2 weeks before and throughout the study. Each subject was asked, to take 5g pectin powder (H, P. Bulmer Ltd., Hereford) three times daily before meals. This was most easily accomplished if the pectin was mixed with 50 ml of water or orange juice, swallowed quickly before it had gelled, and then washed down with more water. Subjects measured their dose with a scoop which delivered 5g. The dose actually taken was assessed by giving each subject a known quantity of more than sufficient pectin for 1 week, and asking them to return the excess at the end of each week before receiving the next week’s supply. The quantity returned was
weighed. The study
was conducted during a 4-week period. During the initial baseline week stools were collected over a 5-day period for the determination of whole-gut transit-time and wet stool weight. Total serum cholesterol, triglycerides, and apohpoprotein B were measured on two occasions, and serum very low density lipoprotein (V.L.D.L.) cholesterol and triglycerides, and serum low density lipoprotein (L.D.L.) cholesterol and triglycerides once. During the next 3 weeks the men took pectin, Whole-gut transit-times and wet stool weights were determined from further 5-day stool collections during the last week. Total serum cholesterol, triglyceride, and apolipoprotein B measurements were repeated 2 days, 1 week, 2 weeks, and 3 weeks after starting pectin, and serum V.L.D.L. and L.D.L. lipids were measured at the end of the period on pectin.
Techniques transit-time was estimated by the time taken for of 2barium-impregnated radio-opaque polyethylene pellets (Portex) taken by mouth to appear in the stools’ which were collected for 5 days, weighed, and X-rayed. Lipid and lipoprotein estimations were made on serum from venous blood collected without stasis after an llh fast. Cholesterol and triglycerides in serum and lipoprotein fractions were measured on the Technicon ‘AutoAnalyzer’-cholesterol by a modification of the Liebermann-Burchard reaction,’ and triglycerides by the standard semi-automated Technicon method.’ Serum v.L.D.L. and L.D.L. were obtained by ultracentrifugation. Freshly collected serum was ultracentrifuged at 100 000 g for 24 h which yielded V.L.D.L. as the supernatant. This was followed by a further 24 h ultracentrifugation of the infranatant, the density of which had been adjusted to 1.063 g/ml. by the addition of a calculated amount of sodium-chloride/sodium-bromide solution, when L.D.L. was obtained as the supernatant.7 Serum-apolipoprotein-B was determined by a doubleantibody radioimmunoassay8 with a highly specific sheep antiserum raised against human lipoprotein of density 1.040-1.053 g/ml, in which apolipoprotein B is the onfv antigen present.9 Results obtained before and during the time subjects were taking pectin were compared by Student’s paired
Whole-gut
80%
or more
t test.
Results Total Serum-lipids
Serum-cholesterol was lower in eleven out of twelve of the men when they were taking pectin than during the initial week of the study. The mean reduction for the was 0-48±0-18 mmol/1 (mean±S.E.M.)—i.e., 7.9±2.6%. The reduction was significant within 1 week
group
of starting
pectin (see accompanying figure). glycerides did not change significantly.
Serum-tri-
Serum-apolipoprotein-B Initial serum-apolipoprotein-B concentrations were ir the normal range for our laboratory (0.60-1.30 g with the exception of one man in whom serum-apolipoprotein-B was raised at 1.86 g/l. His serum-cholesterol
,
i
original pepsin-trypsin digest. That the lymphocyte reactivity is restricted to cceliac patients suggests immunological sensitisation. This may result from an inin the
herent
Coeliac
General medical
patients
Gastroenterology patients
patients Fig, +-Stimulation index
of
lymphocytes
cultured for 48 h with P.H.A.
or
acquired difference in the immune
response to
antigenic determinants of gluten. Alternatively it may result from the stimulation of immunocompetent cells in the jejunal mucosa by antigens that remain after the incomplete degradation of gluten by cœliac mucosa. The ability of a2 to cause an enteropathy in patients suggests that lymphocyte sensitisation may have a role in the pathogenesis of the disease. Indeed, it is known that T lymphocytes have a role in the production of other forms of villous atrophy. Thus studies on animal models, including mouse intestinal allograft rejection," and the colonization of mouse jejunum by the nematode Nippostrongylus braziliensis12 show that villous atrophy, similar to that found in coeliac patients, does not occur in mice deprived of T lymphocytes by thymectomy. Further work is necessary to determine the usefulness of the assay as a screening test for coeliac disease and to attempt to purify further the active component ofB2. We thank Dr W. Jacobson for suggesting these experiments; Dr Max Perutz for initiating our collaboration; Prof. E. Lennox and Dr T. Pearson for helpful advice; and Dr A. P. Dick and Dr J. Hunter for allowing us to study their patients. We thank the M.R.C. for financial help including a Trainee Fellowship for K.S. Requests for reprints should be addressed to K. S., M.R.C. Laboratory of Molecular Biology, University Postgraduate Medical School, Hills Road, Cambridge CB2 2QH. REFERENCES
zer’s fraction
fraction
reproducibly stimulated D,S.A. synthesis in lymphocytes from coeliac patients. However, a subfraction, B2, prepared by sephadex G-50 gel filtration consistently stimulated while Bland B3 did not do so. A dose-response titration using this B2 subfraction is shown in fig. 1. Maximum stimulation was ill nor
B
obtained with 10 µg/ml final concentration of this material. A time-course study (using 10 µg/ml B2) showed peak stimulation at 48 h (fig. 2). Lymphocytes from 4 cœliac patients) which responded well to B2 did not respond to other dietary proteins including &agr;-lactalbumin, &bgr;-lactoglobulin, and casein (data not shown). The response of lymphocytes from a series of cceliac patients and controls to B2 was then measured and is shown in fig. 3. Lymphocyte stimulation by P.H.A. (a potent T-cell mitogen) was measured concurrently (fig. 4. There is a clear difference in the reactivity of the lymphocytes from coeliac patients to B2 when compared with ooth general medical and gastroenterological controls P 001 in both cases). There is no significant difference between any of these groups in the degree of lymphocyte rssponsueness to P.H.A. at 48 h. The various subfractions are at present being tested for their damaging properties by feeding experiments with cceliac volunteers followed by peroral jejunal nopsy, Preliminary results show that B2 and B3 cause istological abnormalities in jejunal mucosa whilst Bl1
joes not.9 Discussion
.
It is clear that lymphocytes from coeliac patients are ’.imutated by the B2 subfraction of gluten whereas lymphocytes from the control groups are not. It may be that ,. preparation of the B2 subfraction concentrates the stimulatory component or removes an inhibitor present
1. 2. 3.
Shiner, M. Gut, 1973, 14, 1. Marsh, M. N. ibid. 1975, 16, 665. Keuning, J. J., Pena, J., Van Leeuwen, A., Van Hoof, J. P., Van Rood, J. J. Lancet, 1976, i, 506. 4. Holmes, G. K. T., Asquith, P., Cooke, W. T. Gut, 1972, 13, 324. 5. Holmes, G. K. T., Asquith, P., Cooke, W. T. Clin. exp. Immun. 1976, 24, 259. 6. Morganroth, J., Watson, D., French, A. B. J. dig. Dis. 1972, 17, 205. 7. Frazer, A. C., Fletcher, R. F., Ross, A., Shaw, B., Sammons, H. G., Schneider, R. Lancet, 1959, u, 252. 8. Dissayanake, A. S., Jerrome, D. W., Offord, R. E., Truelove, S. C., Whitehead, R. Gut, 1974, 15, 931. 9. Anand, B. S., Piris, J., Offord, R. E., Truelove, S. C. Unpublished. 10. Hartzmann, R. H. Cell. Immun. 1972, 4, 127. 11. Macdonald, T. T., Ferguson, A. Gut, 1976, 17, 81. 12. Ferguson, A., Jarrett, E. E. E. ibid. 1975, 16, 114.
COMPARISON OF VITAMIN D AND 25-HYDROXY-VITAMIN-D IN THE THERAPY OF PRIMARY BILIARY CIRRHOSIS* B. WAGONFELD MERRY BOLT JAMES L. BOYER
JAMES
Section
BERNARD A. NEMCHAUSKY JEAN VANDER HORST IRWIN H. ROSENBERG
of Gastroenterology and Liver Study Unit, University of Chicago, Chicago, Illinois 60637, U.S.A.
Skeletal demineralisation and low serum concentrations of 25-hydroxy-vitamin-D were observed in patients with primary biliary cirrhosis. Neither oral nor parenteral vitamin-D increased
Summary
25-hydroxy-vitamin-D
in
serum
or
prevented further 25-hydroxy-
skeletal demineralisation. In contrast, oral
was presented in part at the annual meeting of the AmeriAssociation for the Study of Liver Disease, November, 1975.
*This paper can
392 vitamin-D increased serum-25-hydroxy-vitamin D concentrations in all patients, and bone mineral content either improved or stabilised in all but one. 25-hydroxyvitamin-D may be the preferred form of vitamin-D therapy in primary biliary cirrhosis. Introduction To function physiologically vitamin D must be hydroxylated in the liver to 25-hydroxy-vitamin-D’ and sub-
sequently by the kidney to lx, 25-dihydroxy-vitamin-D.2 Therefore defective vitamin-D utilisation and bone disease might be expected as a complication of either chronic hepatic or chronic renal disease. Bone disease associated with renal disease can be corrected by therapy with 1&agr;, 25-dihydroxy-vitamin-D.34 While osteoporosis and osteomalacia occur in association with chronic hepatic disease, the metabolic basis for this complication has not been clarified.56 Primary biliary cirrhosis, a disease of unknown aetiology characterised by severe derangement of hepatic excretory function and progressive cirrhosis, is commonly complicated by skeletal demineralisation which often progresses despite therapy with high doses of oral or parenteral vitamin D. 7-12 Malabsorption of vitamin D has been found in primary biliary cirrhosis.13 However, the failure of parenteral vitamin D to regularly improve either skeletal demineralisation or calcium malabsorption7-12 suggests that a defect in hepatic metabolism of vitamin D rather than intestinal malabsorption may be the major cause of the skeletal complications of primary biliary cirrhosis. The recently synthesised hepatic metabolite of vitamin D, 25-hydroxy-vitamin-D14 might circumvent such a defect in hepatic metabolism. We therefore determined serum-25-hydroxy-vitamin-D and bone mineral content in primary biliary cirrhosis and compared the therapeutic effect of oral and parenteral vitamin D with that of 25-hydroxy-vitamin-D.
in the within 2%."
ment
same
individual
on
repeated determinationsr
Serum-25-hydroxy-vitamin-D levels were determined by th, competitive protein-binding method of Haddad and Chyu’ with the following modifications. Kidney cytosol binder was prepared from non-rachitic rats. The specific activity of the labelled 25-hydroxy-vitamin-D was 29 Ci/mmole. After sili, cic-acid chromatography, samples were dissolved in ethanol so that aliquots of 50 and 100 µl could be taken for the final binding step. This modification reduced the error mtroduced b evaporation. Improved agreement between duplicates was obtained when the samples were kept in ice during binding and after the addition of dextran-coated charcoal. Samples froma serum pool were reassayed during each run and a mean difference of ±8.1% was obtained. When nine duplicate serumsamples were analysed on different days the mean difference of paired duplicates was ±98%. In our laboratory the mean serum concentration of 25-hydroxy-vitamin-D in seventeen normal volunteers
was
2 3.8 + 5.64 ng/ml.
Patients and Methods
Eight female patients aged 35-62 years were included in study. The patients had had symptoms of primary biliary cirrhosis for 15-125 months at the start of therapy. Seven patients had positive tests for antimitochondrial antibody. Five were clinically jaundiced and had steatorrhcea greater than lOg a day. Only three had advanced cirrhosis morphologically. this
All but one had entered the menopause at least 5 years before the onset of the study. Two patients had severe bone pain. Skeletal mineralisation was assessed in each patient by two criteria: (1) metacarpal cortical thickness, (2) proton-beam absorption. Fine-detail radiographs of the hand were obtained using a fine-grain industrial film (Kodak type M) and were reviewed with optical magnification at 4-10 times by the method of Genant.15 The combined cortical thickness of the left second metacarpal was determined using the technique of Garn.16 Bone mineral content was determined by the technique developed and standardised by Lanzl and Strandjord.17 This method consists of measuring the transmission of a narrow colimated beam of radiation which emanates from an iodine-125 source and is passed through the diaphysis of the middle phalynx of the third digit of the left hand. The transmitted beam is detected by a sodium-iodine scintillation crystal connected to a photo multiplier, discriminator, and scaler. The result is expressed as the linear absorption coefficient,bone, cm-i, after adjustment for bone width and correction for soft tissue and is a measure of the mineral content of both cortical and medullary phalangeal bone. Normal values for our institution have previously been determined from a study of three hundred controls. The reproducibility of the measure-
Summary of the
response to treatment in all
patients.
panel A, therapy with oral vitamin-D is shown in solid lines ai. therapy with parenteral vitamin-D in broken lines. Only those patient In
who had pre and post treatment values are included. Panel B shi,1 the change in linear absorption coefficient of bone expressed as a K cent of age and sex matched control values during vitamin-D therap Panel c shows the response in serum-25-hydroxy-vitamin-D follo therapy with oral 25-hydroxy-vitamin-D. Panel D shows the change’ linear absorption coefficient of bone, expressed as a percent of age sex matched control values after 3-6 months therapy with
25-hydroxy-vitanun-D.
393 25-HYDROXY-VITAMIN-D AND SKELETAL MINERALISATION (MEAN i S.D.) IN PRIMARY BILIARY CIRRHOSIS
CIRCULATING
signs, symptoms, or laboratory data consistent with hypervitaminosis D, doses were subsequently adjusted to maintain circulating vitamin D levels near the control range. On this regimen the linear absorption coefficient of bone either increased
or
remained stable in all but
one
patient (panel D). Discussion Control values for 25-hydroxy-vitamin-D were obtained from normal volunteers ’n late wmter. All 6 patients were studied before any vitamm-D therapy. ’age, sex, and race matched control values for metacarpal cortical thickness 16 were obtained from the data of Garn. sex matched for linear absorption coefficient were control values Ageand 17 obtamedfrom the data of Lanzl and Strandjord. Bumbers in parentheses are the number of patients studied.
Results
Results of the
measurement
of
metacarpal
cortical
thickness, linear absorption coefficients, and serum-25-
hydroxy-vitamin-D for the patients with primary biliary cirrhosis are shown in the accompanying table and are contrasted with the values for the healthy age and sex matched controls. The patients had a mean cortical thickness and mean absorption coefficient significantly less than those of the age and sex matched controls (Student’s t test). In six untreated patients with primary biliary cirrhosis the mean serum-25-hydroxy-vitamin-D level was 4.4±3.4 ng/ml, significantly less than the normal volunteers (23.815.6 ng/ml) (Student’s t test). All patients’ values were below the lowest normal value and two patients had levels unmeasurable by our technique (< 2
neml). At the onset of the study linear absorption coefficient of bone was normal for age and sex. During the 3 months of therapy with parenteral vitamin D, the linear absorption coefficient of bone decreased 10%, a decrease far greater than would be expected by assay variability or the normal aging process. After oral 25-hydroxy-vitamin-D therapy for 3 months, the linear absorption coefficient increased by 5%, a change greater than the variability of the measurement. The response to treatment is summarised in the accompanying figure. Seven patients were treated for 3-6 months with either daily oral vitamin D in doses from 1000 to 50 000 units or monthly parenteral vitamin D 100 000 to 500 000 units subcutaneously. As shown in panel A, none of the six patients in whom both pre and post treatment samples were available responded to either oral or parenteral vitamin D with an elevation in the serum level of 25-hydroxy-vitamin-D to control range. One patient not shown in panel A because her pretreatment concentration was not available did have a normal serum-25-hydroxy-vitamin-D after parenteral vitamin-D therapy. At the end of the 3-6 month treatment period, the linear absorption coefficient of bone was reassessed in each patient (panel B) and a decrease in bone mineral content greater than 2% occurred in all but 2 patients. One patient showed no change. Only the patient with a normal serum-25-hydroxv-vitamin-D after vitamin-D therapy showed improvement in bone mineral content. In contrast to conventional vitamin D, all patients responded to daily oral therapy of 100-200 µg of 25-25-hydroxy-vitamin-D with a rise in serum-25-hydroxy-vitamin-D to normal or high values (panel c). Although no patient demonstrated
Previous studies of the skeletal complications of biliary cirrhosis have described the occurrence of osteomalacia and osteoporosis,19 intestinal calcium malabsorption,2o and low serum-25-hydroxy-vitamin-D.21Despite well-documented malabsorption of vitamin D in primary biliary cirrhosis13 Ajdukiewicz et al.12 have emphasised that symptomatic bone pain and osteomalacia may progress despite long-term therapy with parenteral vitamin D and have suggested that factors other than malabsorption such as decreased hepatic 25-hydroxylation may be of importance.21 Our studies record low serum-25-hydroxy-vitamin-D in patients with primary biliary cirrhosis and show that either oral or parenteral vitamin D in high doses usually fails to correct this abnormality. Since skeletal demineralisation progressed despite parenteral vitamin-D therapy, but stabilised or improved on oral therapy with 25-hydroxy-vitamin-D, impaired hepatic hydroxylation of vitamin D rather than malabsorption would seem to be the major factor in the failure of conventional vitamin-D therapy in primary biliary cirrhosis. Therapy with pharmacological doses of 25-hydroxy-vitamin-D might be expected to cause much greater rises in serum-25-hydroxy-vitamin-D if malabsorption was not present. However, the inability of doses of vitamin D as high as 12.5mg given parenterally to raise serum-25-hydroxy-vitamin-D in vitamin D deficient patients conclusively demonstrates that failure of hydroxylation is a major cause of low 25-hydroxy-vitamin-D levels in these patients and may be an important factor in the vitamin D unresponsiveness in liver disease. Theoretically, liver disease could effect not only the formation, but also hepatic release of 25-hydroxy-vitamin-D. Also, the patient with liver disease might additionally have a defect in renal hydroxylation or may even have some degree of end organ unresponsiveness. The present study does not exclude these other possibilities. These studies show that oral therapy with 25-hydroxy-vitamin-D may circumvent an abnormality in hepatic metabolism of vitamin D in primary biliary cirrhosis and might prevent or ameliorate the hepatic osteodystrophy which often complicates its course. Treatment with 25-hydroxy-vitamin-D should proceed with care until long-term studies determine whether histologic improvement of skeletal demineralisation is also achieved. We thank Dr
roxy-vitamin-D
John Hinman of the Upjohn Company for the 25-hydused in this study and Ms Kathy Linneman for secre-
tarial assistance. This work was supported in part by U.S.P.H.S. Training Grant AM-05147, General Clinical Research Grant RR-55, and N.I.H. Fellowships AM-05150 (Dr Wagonfeld) and AM-05248 (Dr Nemchausky), and by the Gastrointestinal Research Foundation. Dr Boyer is the recipient of Academic Career Development Award AM-70218 from the Institute of Arthritis, Metabolism, and Digestive Diseases. The bone-density apparatus is maintained under Contract E(11-1)-69 from the United States Energy Research and Development Administration.
References overleaf.
394 EFFECT OF PECTIN ON SERUM LIPIDS AND
LIPOPROTEINS, WHOLE-GUT TRANSIT-TIME, AND STOOL WEIGHT P. N. DURRINGTON C. H. BOLTON
A. P. MANNING M. HARTOG
University of Bristol Department of Medicine, Bristol Royal
Infirmary, Bristol BS2 8HW Pectin (12g daily with meals) was taken by twelve healthy men aged 22-45 (mean 25yr) for 3 weeks. This produced a statistically significant mean decrease in total serum-cholesterol of 0.48±0.18 mmol/l (±S.E.M.)—i.e., 7.9±2.6%. The decrease was largely due to a reduction in serum-lowdensity-lipoprotein-cholesterol of 0.45±0.19 mmol/l accompanied by a fall in serum-apolipoprotein-B of 0.11±0.04 g/l. During pectin administration wet stool weight increased from 150±10 to 186±15 g/24 h. There was no clear change in total serum-triglycerides, serum very low density lipoproteins, or in whole-gut transit-
Summary
time.
Introduction PECTIN is the partially methoxylated polymer of galacturonic acid present in the white inner rind and connective tissue of apples, citrus, and various other fruits. Pectin reduced total serum-cholesterol in healthy individuals when taken orally.1-3 However, the effects of pectin on serum triglycerides and lipoproteins and on intestinal function have not previously been reported.
Methods
Subjects and Design of Study Twelve non-obese, healthy male volunteers aged 22-45 (mean 25yr) gave their informed consent to the study, which had been approved by an ethical committee. All the men maintained their normal diet throughout the investigation and
Ponchon, G., Kennan, A. L., DeLuca, H. F. J. clin. Invest. 1969, 48, 2032. Gray, R., Boyle, I., DeLuca, H. F. Science, 1971, 172, 1232. Brickman, A. S., Coburn, J. W., Norman, A. W. New Engl. J. Med. 1972, 287, 891. 4. Brickman, A. S., Coburn, J. W., Massry, S. G., Norman, A. W. Ann. intern. Med. 1974, 80, 161. 5. Collession, L., Grilliat, J. P., Mathieu, J. Presse méd. 1965, 73, 2571. 6. Meunier, P., Dechavanne, M., Prost, G. Lyon Méd. 1973, 229, 1089. 7. Kehayoglou, A. K., Holdsworth, C. D., Agnew, J. E., Whelton, M. J., Sherlock, S. Lancet, 1968, i, 715. 8. Ahrens, E. H., Jr., Payne, M. A., Kunkel, H. G., Eisenmenger, W. J., Blondheim, S. H. Medicine, 1950, 29, 299. 9. Sherlock, S. Gastroenterology, 1959, 37, 574. 10. Wessler, S., Avioli, L. A. J. Am. med. Ass. 1968, 206, 1285. 11. Avioli, L. A., Haddad, J. G. Metobolism, 1973, 22, 507. 12. Ajdukiewicz, A. B., Agnew, J. E., Byers, P. D., Wills, M. R., Sherlock, S. Gut, 1974, 15, 788. 13. Thompson, G. W., Lewis, B., Booth, C. C. J. clin. Invest. 1966, 45, 94. 14. Blunt, J. W., DeLuca, H. F. Biochemistry, 1969, 8, 671. 15. Genant, H. K., Doi, K., Mall, J. C. Invest. Radiol. 1975, 10, 160. 16. Garn, S. The Earlier Gain and the Later Loss of Cortical Bone in Nutritional Perspective; Springfield, Illinois, 1970. 17. Lanzl, L. H., Strandjord, N. Symposium on Low-energy and Gamma Ray Sources and Application, Chicago, Illinois, October, 1964. 18. Haddad, J. G., Chyu, K. J. J. clin. Endocr. 1971, 33, 992. 19. Atkinson, M., Nordm, B. E. C., Sherlock, S. Q. Jl Med. 1956, 25, 299. 20. Whelton, M. J., Kehayoglou, A. K., Agnew, J. E., Turnberg, L. A., Sherlock, S. Gut, 1971, 12, 978. 21. Ajdukiewicz, A. J., Hamlyn, A. N., Preece, M. A., Ribot, C. A., O’Riordan, J. L. H., Sherlock, S. Digestion, 1974, 10, 332. 1. 2. 3.
avoided excessive alcohol intake. No medication was taken for at least 2 weeks before and throughout the study. Each subject was asked, to take 5g pectin powder (H, P. Bulmer Ltd., Hereford) three times daily before meals. This was most easily accomplished if the pectin was mixed with 50 ml of water or orange juice, swallowed quickly before it had gelled, and then washed down with more water. Subjects measured their dose with a scoop which delivered 5g. The dose actually taken was assessed by giving each subject a known quantity of more than sufficient pectin for 1 week, and asking them to return the excess at the end of each week before receiving the next week’s supply. The quantity returned was
weighed. The study
was conducted during a 4-week period. During the initial baseline week stools were collected over a 5-day period for the determination of whole-gut transit-time and wet stool weight. Total serum cholesterol, triglycerides, and apohpoprotein B were measured on two occasions, and serum very low density lipoprotein (V.L.D.L.) cholesterol and triglycerides, and serum low density lipoprotein (L.D.L.) cholesterol and triglycerides once. During the next 3 weeks the men took pectin, Whole-gut transit-times and wet stool weights were determined from further 5-day stool collections during the last week. Total serum cholesterol, triglyceride, and apolipoprotein B measurements were repeated 2 days, 1 week, 2 weeks, and 3 weeks after starting pectin, and serum V.L.D.L. and L.D.L. lipids were measured at the end of the period on pectin.
Techniques transit-time was estimated by the time taken for of 2barium-impregnated radio-opaque polyethylene pellets (Portex) taken by mouth to appear in the stools’ which were collected for 5 days, weighed, and X-rayed. Lipid and lipoprotein estimations were made on serum from venous blood collected without stasis after an llh fast. Cholesterol and triglycerides in serum and lipoprotein fractions were measured on the Technicon ‘AutoAnalyzer’-cholesterol by a modification of the Liebermann-Burchard reaction,’ and triglycerides by the standard semi-automated Technicon method.’ Serum v.L.D.L. and L.D.L. were obtained by ultracentrifugation. Freshly collected serum was ultracentrifuged at 100 000 g for 24 h which yielded V.L.D.L. as the supernatant. This was followed by a further 24 h ultracentrifugation of the infranatant, the density of which had been adjusted to 1.063 g/ml. by the addition of a calculated amount of sodium-chloride/sodium-bromide solution, when L.D.L. was obtained as the supernatant.7 Serum-apolipoprotein-B was determined by a doubleantibody radioimmunoassay8 with a highly specific sheep antiserum raised against human lipoprotein of density 1.040-1.053 g/ml, in which apolipoprotein B is the onfv antigen present.9 Results obtained before and during the time subjects were taking pectin were compared by Student’s paired
Whole-gut
80%
or more
t test.
Results Total Serum-lipids
Serum-cholesterol was lower in eleven out of twelve of the men when they were taking pectin than during the initial week of the study. The mean reduction for the was 0-48±0-18 mmol/1 (mean±S.E.M.)—i.e., 7.9±2.6%. The reduction was significant within 1 week
group
of starting
pectin (see accompanying figure). glycerides did not change significantly.
Serum-tri-
Serum-apolipoprotein-B Initial serum-apolipoprotein-B concentrations were ir the normal range for our laboratory (0.60-1.30 g with the exception of one man in whom serum-apolipoprotein-B was raised at 1.86 g/l. His serum-cholesterol
,
i
