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appearances which were obviously different from those seenin the various forms of glomerulonephritis that were studied.Lack of cross-reactivity between anti-GBM antibody and
Goodpasture’s syndrome and anti-SAP.-Digestion of isolatedGBM with bacterial collagenase released into the supernatantboth the AP-like protein8 and the antigen with which theGoodpasture antibody reacts. However, from 3 h of digestiononwards the AP concentration decreased and after 19 h it wasabsent, whereas the level of the Goodpasture antigen was con-stant. Furthermore, anti-SAP antibody did not compete inthe solid-phase radiometric immunoassay for anti-GBM anti-body. Serum levels of SAP were normal in patients withGoodpasture’s syndrome and their serum did not precipitatein gel with SAP isolated from normal subjects.
DiscussionThe GBM is a complex structure composed largely oftype-
IV collagen linked by various covalent bonds to severalhitherto poorly characterised matrix glycoproteins.15 It is
highly insoluble under physiological conditions and,together with the endothelial and epithelial cell layers, formsthe filtration barrier between the plasma and the urinaryspace. Structural and/or functional abnormalities of thisbarrier are important features of many renal diseases andwhereas the histological, ultrastructural, and immunologicalconcomitants of these abnormalities have been extensivelystudied, little is known about the intrinsic biochemical con-stituents of GBM in disease. We have recently shown that aprotein which reacts identically with serum amyloid P-com-ponent (SAP) in immunoprecipitation tests,8 and whichdespite an earlier report is unrelated to collagen,’-9 is
covalently bound as an intrinsic matrix protein of humanGBM. This enables us to investigate changes within theGBM itself.Direct immunofluorescent staining of renal tissue with
specific anti-SAP antibodies revealed a series of distinctabnormal patterns in different renal diseases. This extends
previous immunohistochemical studies with heterologousantisera to constituents of glomeruli.16-18 Recognition ofthese patterns should help in routine pathological diagnosis,enhancing the information given by standard histological,electron microscopic, and immunological procedures.Staining with anti-SAP may even detect more subtle abnor-malities that are not revealed by the usual methods.
Moreover, these abnormalities may be of pathophysiologicalsignificance. For example, there were considerable abnor-malities in the intensity of staining with anti-SAP of GBM inpatients with minimal-change glomerulonephritis. Thisobservation needs to be confirmed by more extensivestudies, but together with our other findings it suggests thatchanges related to the basement membrane AP may be closelyassociated with changes in the filtration function of theGBM.Functional impairment of the GBM is a feature of Good-
pasture’s syndrome, in which there is an autoclastic autoanti-body directed against an as yet unidentified matrix protein ofGBM.’4 We have shown here that there is no immuno-chemical cross-reactivity between AP and the Goodpasture’santigen.We thank Prof. D. K. Peters for his helpful discussions and permission to
study his patients; Mr J. Dawwas for cutting cryostat sections; Miss SusanMelrose for EM studies; and Mr W. Hinkes, Department of Medical Illus-tration, R.P.M.S., for printing the photomicrographs. This work was
supported by the National Kidney Research Fund and by MRC ProgrammeGrant G979/51 to M.B.P. R.F.D. was supported by the Canadian MRC(present address: Department of Medicine, University Hospital, Saskatoon,Canada).
Requests for reprints should be addressed to M. B. P., ImmunologicalMedicine Unit, Department of Medicine, Royal Postgraduate MedicalSchool, Hammersmith Hospital, Du Cane Road, London W 12 OHS.
REFERENCES
1. Cohen AS, Franklin EC, Glenner GG, Natvig JB, Osserman EF, Wegelius O. In:Wegelius O, Pasternack A, eds. Nomenclature in amyloidosis. London: AcademicPress, 1976: ix.
2. Cathcart ES, Comerford FR, Cohen AS. Immunologic studies on a protein extractedfrom human secondary amyloid. N Engl J Med 1965; 272: 143-46.
3. Bladen HA, Nylen MU, Glenner GG. The ultrastructure of human amyloid asrevealed by the negative staining technique. J Ultrastruct Res 1966; 14: 449-59.
4. Cathcart ES, Skinner M, Cohen AS. Immunogenicity of amyloid. Immunology 1971;20: 945-54.
5. Westermark P, Skinner M, Cohen AS. The P-component of amyloid of human islets ofLangerhans. Scand J Immunol 1975; 4: 95-97.
6. Breathnach S, Black MM, Dyck RF, Pepys MB. Unpublished.7. Dyck RF, Kershaw M, McHugh N, Pepys MB. Immunohistochemical staining of
normal and pathological human tissues with antibody to serum amyloidP-component (SAP). In: Glenner GG, Pmho e Costa P, de Freitas F, eds. Amyloidand amyloidosis. Amsterdam:Excerpta Medica, 1980: 50-54.
8. Dyck RF, Kershaw M, McHugh N, Lockwood CM, Duance V, Baltz ML, Pepys MB.Amyloid P-component, a constituent of normal human basement membrane. J ExpMed (in press).
9. Schneider H-M, Loos M. Amyloid P-component—a special type of collagen? VirchowsArch B Cell Path 1978; 29: 225-28
10. Pepys MB, Dash AC, Munn EA, et al. Isolation of amyloid P-component (protein AP)from normal serum as a calcium-dependent binding protein Lancet 1977; i:
1029-31.
11. Goding JW. Conjugation of antibodies with fluorochromes: modifications to thestandard methods. J Immunol Methods 1976; 13: 215-26.
12. Johnson GD, Holborow EJ, Dorling J. Immunofluorescence and immunoenzymetechniques. In: Weir DM, ed. Handbook of experimental immunology. Oxford:Blackwell Scientific Publications, 1978: 15.1.
13. Coleman DV, Russell WJI, Hodgson J, Tun PE, Mowbray JF. Human papovarius inPap smears of urinary sediment detected by transmission electron microscopy. JClin Path 1977; 30: 1015-20.
14. Lockwood CM, Amos N, Peters DK. Goodpasture’s syndrome: radioimmunoassay formeasurement of circulating anti-GBM antibodies Kidney Int 1979; 16: 93(A).
15. Kefalides NA, ed. Biology and chemistry of basement membranes. New York:Academic Press, 1978.
16. Scheinman JI, Fish AJ, Michael AF. The immunohistopathology of glomerularantigens, II. The glomerular basement membrane, collagen, and actomyosinantigens in normal and diseased kidneys. J Lab Clin Med 1974; 54: 1144-54.
17. Petterssen EE, Colvin RB. Cold insoluble globulin (fibronectin, LETS protein) innormal and diseased human glomeruli: Papain-sensitive attachment to normalglomeruli and deposition in crescents. Clin Immunol Immunopath 1978; 11:
425-36.
18. Scheinman JI, Foidart JM, Gehron-Robey P, Fish AJ, Michael AF. The immunohis-tology of glomerular antigens. IV Laminin, a defined noncollagen basement mem-brane glycoprotem. Clin Immunol Immunopath 1980, 15: 175-89.
COMPARISON OF THE EFFECTS OF REGULARAND ENTERIC-COATED ASPIRIN ONGASTRODUODENAL MUCOSA OF MAN
JAMES W. HOFTIEZERMARLYN BURKS
GERALD R. SILVOSOKEVIN J. IVEY
Department of Medicine, Harry S. Truman Memorial VeteransHospital and University of Missouri School of Medicine, Columbia,Missouri; and University of California, Irvine Ca 92649, California,
U.S.A.
Summary To determine whether the topical or sys-temic effects of aspirin are of greater impor-
tance in the production of gastroduodenal mucosal damage,the effects of regular and enteric-coated aspirin were com-pared in 9 healthy volunteers in a 2-week crossover endo-scopic study. All subjects developed multiple gastric erosionswhile taking regular aspirin; 2 subjects developed one gastricerosion each while taking enteric-coated aspirin. 5 subjectsdeveloped duodenal erosions while taking regular aspirin,whereas none developed an erosion while taking enteric-coated aspirin: Mean fasting salicylate levels were similar inthe two groups. It is concluded that regular aspirin causes agreater amount of gastroduodenal mucosal damage than does
610
enteric-coat aspirin despite similar serum-salicylate levels.This suggests that the topical effects of aspirin are of greaterimportance than the systemic effects in the production ofgastroduodenal mucosal damage in healthy subjects.
Introduction
ORDINARY aspirin is widely believed to cause gastricmucosal damage as shown by a tendency to reduce the gastricmucosal potential difference, to induce occult bleeding, andto cause erosions and ulcers. In a study of 82 patients on long-term aspirin therapy we found gastric ulcers in 28% ofpatients taking non-enteric-coated aspirin and in 6% ofpatients taking enteric-coated aspirin (p<0’05), with no
significant difference in serum-salicylate levels between thetwo groups. If enteric-coated aspirin can be shown toproduce less gastrointestinal damage than regular aspirin, asignificant reduction in morbidity without a compromise ineffectiveness might be achieved by switching patients withindications for long-term aspirin treatment to enteric-coatedforms. We have compared the effects of enteric-coated aspirin(’Ecotrin’) versus regular aspirin (Bayer) taken regularly for 2weeks on the gastroduodenal mucosa of healthy volunteers.
Methods
Subjects9 healthy volunteers aged 21 to 31 took part in the study. They
had no history of gastrointestinal symptoms, aspirin intolerance, orulcer disease. All had normal physical examination, gastroduodeno-scopy, and laboratory tests (an automated screening for blood ureanitrogen, serum glutamic oxaloacetic transaminase, serum
bilirubin, serum Na, Ka, Cl-, serum cholesterol, and serumalkaline phosphatase; other electrolytes; complete and differentialblood counts; prothrombin time; activated partial thromboplastintime; urinalysis; and stool guaiac test) before entry into the study.All agreed to abstain from alcohol or other medications during thestudy.
Study Plan .
The study, which was approved by the Institutional Human UseCommittee, was a single-blind (investigator) crossover study, con-sisting of three consecutive 2-week periods. In the first and thirdperiods, each volunteer took 2 tablets of either-regular or enteric-coated aspirin, with each of three meals and at 10.30 P.M.-a total of8 tablets (2 - 4 g) of aspirin per day. The order in which they took thetwo types of aspirin was randomly determined. In the middle(washout) period no medications of any kind were taken.At the end of each 2-week period, the subjects had a history taken,
and underwent a physical examination, gastroduodenoscopy, andlaboratory tests (the automated screening tests, complete and differ-ential blood counts, and stool guaiac test). Blood samples for fastingserum-salicylate levels were drawn every week, at 8 A.M.
Technical AspectsThe history was taken on a standardised form, which covered an
extensive range of upper-gastrointestinal symptoms. A positiveTABLE I-ENDOSCOPY GRADING SCALE
TABLE II-COMPARISON OF EROSIONS, SALICYLATE LEVELS, AND
SYMPTOMS ASSOCIATED WITH REGULAR AND
ENTERIC-COATED ASPIRIN
Sal=Fasting serum-salicylate level.
symptom history during each 2-week study period, regardless ofduration or frequency, was graded 1, and a negative history wasgraded 0, for statistical analysis.For gastroduodenoscopy an Olympus GIF P-2 pasdiatric endo-
scope (Olympus Corporation of America, New Hyde Park, NewYork) was used. Each subject came to endoscopy having fasted after
’ the last dose of aspirin, which was taken at 10.30 P.M the previousevening. The fundus, antrum, and duodenum were each gradedseparately, the lesions being classified and graded according to tableI. Statistical analysis was applied to the resulting grades. Each typeof lesion was analysed separately for each anatomical area. Each areawas graded before the endoscope was advanced through the area, toprevent trauma artefact. Any white fibrin-based mucosal lesion,regardless of size, was designated an erosion, since the distinctionbetween erosion and ulceration requires a biopsy.The serum from the weekly fasting blood samples was frozen and
stored until the end of the study, when the salicylate levels weredetermined in a single run. The method used was a modification ofthe spectrofluorometric procedure of Bender for quantification ofserum salicylates.2
Statistics
All data, graded as described above, were analysed with theWilcoxon signed rank test for paired populations.
Results
Endoscopic FindingsGastric erosions.-No subject had erosions at the initial
endoscopy. All 9 subjects studied developed multiple antralerosions while taking regular aspirin (p<0’005 comparedwith the baseline endoscopy). The number of lesions variedfrom 3 to more than 50 (table n), with a mean of 14. Thelargest of these lesions was 0’5 5 cm in diameter. One subjecthad active oozing of blood from erosions at the time of endo-
TABLE III-COMPARISON OF ERYTHEMA GRADES ASSOCIATED WITHREGULAR AND ENTERIC-COATED ASPIRIN
611
scopy. While taking enteric-coated aspirin, 2 subjects deve-loped 1 erosion each (p<0-005 compared with regularaspirin). All erosions healed by the end of the 2-week washoutperiod, without specific therapy. No subject developed gas-tric fundic erosions.Duodenal erosions.-No subject had erosions at the
screening endoscopy. 5 developed multiple duodenal-bulberosions while receiving regular aspirin. No subject had duo-denal erosions after taking the enteric-coated aspirin(p<0-025). The mean number of erosions in subjects whodeveloped them was 9. All erosions had healed within 2
weeks, without specific therapy.Gastric erythema.-l subject had mild antral erythema at
the time of the screening endoscopy. All 9 subjects developeddiffuse antral erythema (4 +, table III), while taking regularaspirin. 4 had some degree of antral erythema after taking theenteric-coated aspirin, but in only 1 case did this represent aprogression from the time of the preceding endoscopy (i.e.,baseline or washout period), and in no case did the severityafter the enteric-coated aspirin exceed that found after regularaspirin (p<0-005). 3 subjects developed diffuse 4+ fundalerythema while taking regular aspirin, and 1 subject had 2 +fundal erythema after the enteric-coated aspirin (p>0 05). 3subjects had residual antral erythema after the 2-weekwashout period between the drug challenges.Duodenal erythema.- No subject had duodenal erythema at
the screening endoscopy. 6 subjects developed diffuse duo-denal erythema while taking regular aspirin, and 3 subjectshad a milder degree of duodenal erythema after the enteric-coated aspirin (p<0 -01, table III).
SymptomsNo-subject had gastrointestinal symptoms at the beginning
of the study or during the washout period. 6 of the 9 deve-loped gastrointestinal symptoms while taking regular aspirin(table II). 4 subjects had burning epigastric pains relieved byeating, 1 had bloating upper-abdominal pains, and 1 subject,during his last week of regular aspirin, was awakened fourtimes between 12 30 and 1 30 A.M. by hunger pains, whichwere relieved by eating. This subject (no. 1) was the only onewho developed symptoms (late-evening hunger pains) whiletaking enteric-coated aspirin (p<0 . 025 compared with
regular aspirin). This subject had experienced burning epi-. gastric pains while taking regular aspirin.
Salicylate LevelsThe average fasting serum-salicylate levels 10 h after the
, last dose was 7 4±SE 1 4 mg/dl for regular aspirin and7.8±1.5 mg/dl for enteric-coated aspirin. There was nocorrelation between salicylate levels and the amount of
gastroduodenal-mucosal damage, either between persons orin the same person between studies (table II).
Laboratory Data
No subject had abnormal blood values, and none developedany abnormalities during the study. The stool-guaiac tests,done on ’Hemoccult’ cards, were all negative.
Additional Results
Subject 4 underwent a third 2-week study period duringwhich he took 3 enteric-coated aspirin tablets four times a day(12 tablets or 3.6 6 g aspirin). During this period his averagefasting salicylate level was 18’8 mg/dl, compared with 7’9
mg/dl on 8 regular aspirin tablets a day and 6’ 3 mg/dl on 8enteric-coated tablets per day. After 2 weeks endoscopyshowed diffuse antral erythema with no erosions and anormal duodenum.
Subject 6 had salicylate levels estimated at various timesthroughout the 24 h period before the end of the 2-week studyperiods for both drugs, to determine whether the salicylatelevels fluctuated during the day. The salicylate levels in thissubject were 27 mg/dl, 26 mg/dl, and 24 mg/dl for fasting, 2 h,and 4 h after a dose of regular aspirin, and 25 mg/dl, 24 mg/dl,and 28 mg/dl for the same times after enteric-coated aspirin.This demonstrates a consistent blood level of salicylate forthis patient throughout the day during both types of aspirinuse.
Discussion
Our study shows a significant increase in gastroduodenalmucosal lesions after 2 weeks of regular aspirin therapy. Webelieve this is the first study to emphasise the high incidenceof gastric as well as duodenal erosions after acute aspirinadministration. We are unaware of any previous controlledcrossover endoscopic study comparing the effects of regularand enteric-coated aspirin on gastroduodenal mucosa. Tocause damage to the gastric mucosa, aspirin must penetratethe cell. Gastric acid also seems to be necessary for aspirin toinduce injury to the gastric mucosa.4,5 At low pH, around itspKa of 3’ 5, aspirin can induce lesions in the human smallbowel with or without added acid.6 The acidity of thestomach favours topical aspirin damage, but most of the smallintestine (pH 7) is sufficiently buffered to minimise thiseffect. The duodenum, however, is often at a pH of 2-4,7,8which could result in aspirin damage. Enteric-coated aspirintablets are designed to break down in the small intestine at apH of 6 to 7. Thus, theoretically, they bypass the stomach andin general the duodenum, without producing damage in theseareas. This proved to be so in the present study.We have previously reported that patients with rheumatic
diseases receiving long-term aspirin therapy have a highprevalence of duodenal lesions (4% having ulcers, 14%erosions, and 32% erythema in the duodenum).9 The preva-lence of duodenal erosions was similar, however, whetherpatients were taking regular or enteric-coated aspirin.Some studies in animals suggest that parenteral aspirin can
produce gastric lesions similar to those produced with oralaspirin.10-12 Thus, gastric-mucosal damage might occur inman, regardless of how aspirin is administered, through asystemic effect.13,14 Our study, however, demonstrates asignificantly lower incidence of gastroduodenal-mucosaldamage with enteric-coated aspirin than with regular aspirin.Average serum-salicylate levels were not significantlydifferent between the regular and enteric-coated aspiringroups, and there was no intrasubject correlation betweensalicylate levels and gastroduodenal-mucosal damage. Theformation of a single erosion in 2 of the 9 subjects takingenteric-coated aspirin could be due to an occasional enteric-coated tablet dissolving in the stomach. Another possibility isreflux of aspirin, released in the duodenum, into the stomach.Duodenogastric reflux is common in normal subjects. 15-17
6 subjects developed upper-gastrointestinal symptomswhile taking regular aspirin, compared with only 1 takingenteric-coated aspirin. Several subjects with lesions,however, did not have symptoms. Symptoms were intermit-tent in the subjects who had them and were never presentconstantly. The results of our study have important implica-tions for the large group of patients taking regular aspirin,
612
particularly for periods of several weeks. A prospective long-term crossover study is now underway in patients requiringchronic aspirin therapy.We thank Dr John Hewett for the statistical analyses. This work was
supported in part by the Medical Research Service of the Veterans Administra-tion, the Clinical Research Center, NIH grant no. RR00287-12, of the Uni-versity of Missouri School of Medicine, and the Gastroenterology ResearchFoundation Inc. of Mid-Missouri.
Requests for reprints should be addressed to K.J.I., Division of Gastro-enterology, Department of Medicine, Long Beach Veterans AdministrationHospital, Long Beach, California 90822, U.S.A.
REFERENCES
1. Silvoso GR, Ivey KJ, Butt JH, et al. Incidence of gastric lesions in patients withrheumatic disease on chronic aspirin therapy. Ann Intern Med 1979; 91: 517-20.
2. Bender GT. Chemical instrumentation: A laboratory manual based on clinical chemi-stry. Philadelphia: WB Saunders Co, 1972: 62-64.
3. Davenport HW. Gastric mucosal injury by fatty and acetylsalicylic acids.
Gastroenterology 1964; 46: 245-53.4. Bowen Bk, Krause WJ, Ivey KJ. Effect of sodium bicarbonate on aspirin-induced
damage and potential difference changes in human gastric mucosa. Br Med J 1977;ii: 1052-55.
5. MacKercher PA, Ivey KJ, Baskin WN, et al. Protective effect of cimetidine on aspirininduced gastric mucosal damage. Ann Intern Med 1977; 87: 676-79.
6. Ivey KJ, Baskin WN, Krause WJ, et al. Effect of aspirin and acid on human jejunalmucosa. An ultrastructural study. Gastroenterology 1979; 76: 50-56.
7. Andersson S, Grossman MI. Profile of pH, pressure, and potential difference at gas-troduodenal junction in man. Gastroenterology 1965; 49: 364-71.
8. Archambault AP, Rovelstad RA, Carlson HC. In situ pH of duodenal bulb contents innormal and duodenal ulcer patients. Gastroenterology 1967; 52: 940-47.
9. Lockard OO, Ivey KJ, Butt JH, et al. The incidence of duodenal lesions in patients withrheumatic diseases on chronic aspinn therapy. Gastrointest Endosc 1980; 26: 5-7.
10. Brodie DA, Hooke HF: Effects of route of administration on the production of gastrichemorrhage in the rat by aspirin and sodium salicylate. Am J Dig Dis 1971; 16:985-89.
11. Brodie DA, Tate CL, Hooke KF. Aspirin and intestinal damage in rats. Science 1970;170: 183-84.
12. Bugat R, Thompson MR, Aures D, et al. Gastric mucosal lesions produced byintravenous infusion of aspirin in cats. Gastroenterology 1976; 71: 754-59.
13. Menguy R. Gastric mucosal injury by aspirin. Gastroenterology 1966; 51: 430-32.14. Grossman MI, Matsumoto KK, Lichter RJ. Fecal blood loss produced by oral and
intravenous administration of various salicylates. Gastroenterology 1961; 40:383-88.
15. Rhodes J, Barnardo DE, Phillips S, et al. Increased reflux of bile into the stomach inpatients with gastric ulcer. Gastroenterology 1969; 57: 241-52.
16. Fisher R, Cohen S. Physiologic charactaeristics of the human pyloric sphincter.Gastroenterology 1973; 64: 67-75.
17. Wormsley KG. Aspects of duodeno-gastric reflux in man. Gut 1972; 13: 243-50.
METABOLITES OF VITAMIN D IN HUMANVITAMIN-D DEFICIENCY:EFFECT OF VITAMIN D3 OR
1,25-DIHYDROXYCHOLECALCIFEROL
S. E. PAPAPOULOS
L. J. FRAHERT. L. CLEMENS
J. GLEEDJ. L. H. O’RIORDAN
Department of Medicine, The Middlesex Hospital, London
Summary Circulating concentrations of hydroxylatedmetabolites of vitamin D were measured in
seven Asian patients with histologically proven osteomalaciabefore and during treatment with either cholecalciferol
(vitamin D3) or 1,25-dihydroxycholecalciferol. All patientsshowed an excellent clinical and biochemical response totreatment irrespective of type of vitamin D administered.Circulating concentrations of 25-hydroxycholecalciferol and24,25, 25,26, and 1,25 dihydroxycholecalciferols were
abnormally low in the untreated patients. In five patientstreated with small doses of cholecalciferol (3000 units, 75 µgdaily) the concentration of 1,25-dihydroxycholecalciferolrose rapidly to normal and reached supra-normal levels
within 72 h. Raised concentrations (up to 200 pg/ml) weresustained for several months and then started falling tonormal. Serum 24,25 and 25,26 dihydroxycholecalciferolsrose only gradually, after circulating 25-hydroxycholecal-ciferol concentration had increased to normal. In contrast, inthe two patients who received 1,25-dihydroxycholecalciferol(1 pg daily) serum concentrations of this metabolite rose tonormal and only occasionally exceeded the upper limit ofnormal. The highest concentration observed was 80 pg/ml.Healing of osteomalacia occurred, however, in these twopatients in the absence of any measurable increases in
25-hydroxycholecalciferol and 24,25 or 25,26 dihydroxy-cholecalciferols which all remained abnormally low. Thus, itseems that 1,25-dihydroxycholecalciferol is the most
important factor for the healing of vitamin-D-deficient osteo-malacia and that other hydroxy metabolites are unimportant.
Introduction
1,25-DIHYDROXYCHOLECALCIFEROL (1,25-DHCC) is
recognised as the most important, if not the only, biologicallyactive form of vitamin D involved in the mobilisation ofcalcium from intestine and bone. In vitamin-D-deficientanimals concentrations of circulating 1,25-DHCC were lowor undetectable, 1,2 and synthetic 1,25-DHCC administeredto such animals, had a potent antirachitic effect.’-5 In man,however, there is controversy about the role of 1,25-DHCCboth in the pathogenesis and cure of vitamin-D-deficientosteomalacia. This has mainly stemmed from reports ofnormal serum concentrations of this metabolite in patientswith osteomalacia due to vitamin-D deficiency6,7 and fromhistological studies8 of such patients during treatment with1,25-DHCC. It has been suggested that in human vitamin-D-deficient osteomalacia metabolites of vitamin D other than1,25-DHCC may be important in the development of thedisease and healing. 7-9
.
To study these problems further, we have followed changesin the circulating concentrations of the hydroxylated meta-bolites of vitamin D before and during treatment with chole-calciferol or 1,25-DHCC in seven Asian patients with osteo-malacia.
Methods
Calcium, phosphate, alkaline phosphatase, and albumin weremeasured in serum by automated techniques on a Vickers M 300autoanalyser. Serum calcium was corrected for albumin binding.(Normal values for adults: calcium 2-20-2-55 mmol/1; phosphate0.6-1.3 3 mmol/1; alkaline phosphatase 20-85 IU/1.) Serum para-thyroid hormone (PTH) concentrations were measured by a
homologous amino-terminal specific immunoradiometnc
assay. 10, 11 The upper limit of normal in serum is 120 pg/ml and the
limit of detection is 40 pg/ml. Serum 25-hydroxycholecalciferol(25-HCC) concentrations were determined by a protein-bindingassay12 (normal range 3-30 ng/ml) and circulating 1,25-DHCC and25,26-dihydroxycholecalciferol (25,26-DHCC) by radioimmuno-assays; 13, normal adult. values: 1,25-DHCC, 22-59 pg/ml,mean±SEM 40±1.9 pg/ml; 25,26-DHCC, 71-423 pg/ml, mean244±27.4 4 pg/ml. Serum 24,25-dihydroxycholecalciferol(24,25-DHCC) was also measured by radioimmunoassay byadapting the method used for the measurement of the other twodihydroxy metabolites. For this assay antiserum 02282 in a dilutionof 1:75000 was used with tritiated 1,25-DHCC as tracer andchemically synthesised 24,25-DHCC as standard. Recovery of themetabolite during extraction of serum samples and sequentialchromatography on Sephadex LH 20 and high-pressure liquidchromatography was monitored by biologically generated 15
tritiated 24,25-DHCC and averaged 50±0 . 8%. The mean concen-
