Abnormal Vitamin D Metabolism in Patients with Cirrhosis GERSHON

W. H E P N E R ,

MD, M A R T I N R O G I N S K Y , MD, and H I N G FAI MOO, PhD

To assess the role of hepatic function and alcohol on vitamin D metabolism, serum 25hydroxyvitamin D (25-OHD) levels were measured in 20 healthy nonalcoholic control subjects, 31 "inactive" cirrhotics whose alcoholism was in remission, 8 alcoholic cirrhotics, and 15 alcoholics with normal liver function. Cirrhosis, but not alcoholism, was associated with low serum 25-OHD levels. The aminopyrine breath test (ABT) was performed because aminopyrine, like vitamin D:~, is metabolized by hepatic microsomes; the A B T correlated highly (r = 0.74, P < 0.01) with serum 25-OHD in the inactive cirrhotics. After an intravenous injection of I20 tzg vitamin Da, serum 25-OHD rose significantly within 24 hr in 6 healthy controls and 2 patients with celiac disease but not in 6 inactive cirrhotics. The data suggest impaired 25-hydroxylation of vitamin-D impaired in patients with cirrhosis, related predominantly to the degree of hepatic dysfunction.

Vitamin D undergoes important biotransformation in the liver (1, 2) and the kidney (3). The hepatic metabolite, 25-hydroxyvitamin D (25-OHD), has biological activity on intestine (4) and bone (5), and is probably the precursor of the renal metabolite of vitamin D, 1,25-dihydroxyvitamin D [1,25-(OH)=,D] (6), which is the most biologically active form of vitamin D that has yet been isolated from m a m m a lian or avian tissues (6-8). Since vitamin D is metabolized both by liver and kidney, abnormal vitamin-D metabolism and bone disease might be expected to be associated with liver disease, by analogy with chronic renal disease, where renal osteodystrophy is commonly recognized (9). An increased incidence of osteoporosis has been described in patients with portal cirrhosis (1012), and also in alcoholics in w h o m no liver dysfunction was recognized (13-15). Since osteoporosis From the Department of Medicine, The Milton S. Hershey Medical Center of The Pennsylvania State University, Hershey, Pennsylvania 17033, and the Division of Endocrinology, Nassau County Medical Center, East Meadow, Long Island, New York 11554.

This work was supported in part by Grant AM 17303 from the National Institutes of Health. Address for reprint requests: Dr. Gershon W. Hepner, Department of Medicine, Harbor General Hospital, Torrance, California 90509. Digestive Diseases, Vol. 2l, No. 7 (July 1976)

may be the first pathological change in the bone in patients with vitamin D deficiency (16), it was decided to study a group of alcoholic and nonalcoholic subjects with and without cirrhosis in order to determine the effect of alcohol and hepatic dysfunction on vitamin D metabolism. F u r t h e r m o r e , since hepatic biotransformation of vitamin D:~ occurs in hepatic m i c r o s o m e s (17), hepatic microsomal function was also studied by the aminopyrine breath test (18) in order to assess the effect of hepatic microsomal function on vitamin D metabolism. MATERIALS AND M E T H O D S Patients Studied All patients were studied as inpatients at the Hershey Medical Center and fell into five categories. There were 20 nonalcoholic control subjects, whose age was 42 + 11 years (mean -+ SD); they included 12 patients with gastrectomy, 4 with peptic esophagitis, 2 with cancer of the colon, and 4 with traumatic fractures. The mean serum 25-OHD in these patients was similar to that of normal controls in our laboratory. There were 31 cirrhotics whose alcoholism was in remission; these inactive cirrhotics are termed "'inactive cirrhotics" and their age was 48 + 8 years. They all had alcoholic cirrhosis diagnosed by liver biopsy and had all stopped drinking alcohol at least 6 months before the study. There were 8 alcoholic

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After fasting blood samples had been taken from 6 nonalcoholic controls, 6 inactive cirrhotics, and the 2 patients with celiac disease, they were given intravenous injection of 120/zg vitamin D:~, dissolved in 1 ml propylene glycol. None of the patients so studied had ascites. Blood samples were taken 24 hr later in all patients so treated, as were further samples for a period of 8 days in 8 of the patients, and were assayed on pre- and posttreatment samples.

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Fig 1. Serum 25OHD levels in patients studied.

cirrhotics, whose age was 37 + 10 years. They, too, had alcoholic cirrhosis diagnosed by liver biopsy. There were 15 alcoholic controls who had normal liver function and whose age Was 43 +- 7 years. The alcoholic patients were all consuming the equivalent of one fifth or more of alcoholic spirits per day. The alcoholic controls were clinically well nourished and none had any clinical, biochemical, or hematological evidence of deficiency of water-soluble vitamins; apart from 6 nonalcoholic cirrhotics who had ascites, all the cirrhotics also seemed well nourished, and the only vitamin deficiency apparent was folic acid deficiency present in 4 of the 8 alcoholic cirrhotics. Vitamin D intake appeared to be similar in the alcoholic controls, nonalcoholic controls, and inactive cirrhotics, but appeared to be decreased in the 4 alcoholic cirrhotics who also had evidence of folic acid deficiency. In addition 2 patients with untreated celiac disease and osteomalacia were studied. No patient had taken drugs known to effect hepatic function for at least one month before the study. Routine assessment of bone x-ray of all the noncirrhotic subjects showed no radiological bony abnormalities. Only one patient with cirrhosis had any clinical evidence of metabolic bone disease. He had a pathological fracture of the humerus and a bone biopsy showed Paget's disease without evidence of osteomalacia. Studies were performed over a period of 18 months. There was no difference in the seasonal distribution of the subjects studied since studies were performed in nonalcoholic controls, inactive cirrhotics, alcoholic cirrhotics, and alcoholic controls with equal frequency during the four seasons.

Serum 25-OHD levels were measured by a modification of the competitive protein-binding assay of Belsey et al (19), as described by Rosen et al (20). This assay utilizes a specific binding protein isolated from rachitic rat serum with a high affinity for 25-OHD. Since reference 25OHD2 was not available to compare its displacement potency with that of 25-OHD:~, the levels of serum 25-OHD reported in this study represent total serum 25-OHD, 25OHD2, as well as 25-OHD:~. Other vitamin D metabolites, including 1,25(OH)~D, are not measured by this assay. Crystalline 25-OHD:~ was used as the reference standard, and the tracer was 26,27-[:~H]25-OHD:~. The sensitivity of the assay is 1 ng/ml and the interassay variability is -+ 8%.

Assessment of liver function In all patients, serum albumin, bilirubin, calcium, magnesium, phosphate, aspartate transaminase, and alkaline phosphatase were measured using a Technicon 12/60 autoanalyzer. These tests were normal in all alcoholic control subjects. The aminopyrine breath test (18) was also performed on all the alcoholic cirrhotic and control subjects as a further assessment of liver function, and was normal in all the controls (> 4.5%); no liver biopsies were performed on these patients.

Statistical Analysis Comparison between patients was performed by means of a Student's t test. In studies comparing serum 25-OHD levels before and after parenteral administration of 120/~g vitamin D:~, a paired t test was performed.

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Experimental Design A sample of fasting blood was taken for each assay of serum 25-OHD. The serum was stored at - 2 0 ~ C until assayed.

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2. Serum 25-OHD in patients with inactive cirrhosis

plotted againsi aminopyrine breath test. Digestive Diseases, Vol. 21, No. 7 (July 1976)

VITAMIN D METABOLISM IN CIRRHOSIS RESULTS Serum 25-OHD levels in the five groups of patients studied are shown in Figure 1. The level in the nonalcoholic controls was 35 + 12 ng/ml (mean -+ so), significantly higher than the inactive cirrhotics (20 + 11 ng/ml, t - 4.35, P < 0.001). Serum 25-OHD was lower in the alcoholic controls (28 + 12 ng/ml) than in the nonalcoholic controls (34 -+ 12 ng/ml) inactive (t = 2.26, P < 0.05). The 25-OHD level in the alcoholic cirrhotics, 12 -+ 9 ng/ ml, was lower than in the inactive cirrhotics, but the difference was not statistically significant (t = 1.93, P > 0.05). There was a highly significant correlation (r = 0.74, P < 0.01) between the percentage of 14C excreted in 14CO2 2 hr after administration of [14C]aminopyrine and serum 25-OHD in the nonalcoholic cirrhotics (Figure 2). Serum albumin also correlated highly with serum 25-OHD in these p a t i e n t s (r = 0.70, P < 0.01, Figure 3). T h e r e was no significant correlation (P > 0.1) between the aminopyrine breath test and serum 25-OHD in the 8 alcoholic cirrhotics or the alcoholic and nonalcoholic controls. The effect of a parenteral dose of 120/xg vitamin D:~ on serum 25-OHD 24 hr after administration of the vitamin is shown, for 6 nonalcoholic controls, 2 celiacs, and 6 nonalcoholic (inactive) cirrhotics, in Figure 4. There was a highly significant rise in serum 25-OHD after administration of the vitamin in the 6 nonalcoholic controls (t = 9.77, P < 0.001), but no significant change in the serum 25-OHD level in the nonalcoholic cirrhotics similarly treated (t = 1.17, P > 0.05). The serum 25-OHD level rose by more than 13 ng/ml in one celiac and by 25 ng/ml in the other. In Table 1, serum 25-OHD is shown 27 days after parenteral vitamin D:~ in 8 subjects. The serum 25-OHD remained elevated in the patients who responded after 24 hr, and remained relatively lower in the 4 inactive cirrhotics.

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controls(26), and both osteomalacia and osteoporosis have been described in these patients (21-24). In portal cirrhosis, malabsorption of fat (27, 28) and presumably of fat-soluble vitamins such as vitamin D occurs, but malabsorption of vitamin D is unlikely to explain all the data in the present study. Our data suggest that cirrhotics have impaired 25-hydroxylation of Vitamin D. First, since vitamin D is hydroxylated in liver microsomes (17), hepatic p a r e n c h y m a l disease might be

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DISCUSSION Previous studies of vitamin D metabolism in patients with hepatobiliary disorders have tended to concentrate on patients with biliary obstruction (21-25). In that situation malabsorption of vitamin D probably occurs, due to decreased secretion of bile acids into the intestinal lumen. Serum 25O H D levels m e a s u r e d in a group of patients with biliary cirrhosis were lower than in a group of healthy Digestive Diseases, Vol. 21, No. Z (July 1976)

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CIRRHOSIS

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Fig 4. Serum 25-OHD in nonalcoholic controls, mactive cirrhotics, and celiacs before and after administration of 120 #g vitamin D:~.

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HEPNER ET AL TABLE

1. S E R U M 2 5 - O H D

AFTER TREATMENT

W I T H P A R E N T E R A L V I T A M I N D a IN 4 S U B J E C T S W I T H O U T WITH INACTIVE CIRRHOSIS

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expected to cause a decrease in serum 25-OHD levels. Secondly, serum 25-OHD levels correlated with indices of hepatic function, serum albumin and the aminopyrine breath test (18), in the nonalcoholic cirrhotics. Thirdly, parenterai injection of a dose of vitamin Da which caused an elevation of serum 25OHD in control subjects failed to cause an elevation of serum 25-OHD in the cirrhotics similarly treated, even 4 days after parenteral vitamin D:~ administration. It is of some interest that D e L u c a (7) failed to show a rise in serum 25-OHD level 48 hr after parenteral injection of vitamin Da in 3 healthy volunteers. It is well recognized that such treatment causes a change of serum parathyroid hormone within 48 hr in patients with vitamin D deficiency (29); it is possible that the effect of vitamin D therapy on serum 25-OHD depends on the pretreatment level of serum 25-OHD and 25-OHD stores and that rises may be seen more readily in patients whose pretreatment levels were low or low normal. The abnormally low serum 25-OHD level in the cirrhotic patients may be due to causes other than hepatic mitochondrial or microsoma! dysfunction. First, hepatic protein-binding of serum 25-OHD may be decreased in cirrhotic patients. This could affect clearance of this substance and thus explain its lower plasma level. Also, since 25-OHD is known to cause feedback inhibition of hepatic 25hydroxylation of vitamin D:~, decreased proteinbound 25-OHD, with a concomitant increase in the free serum 25-OHD, might cause decreased 25-hydroxylation in cirrhotic patients. The fraction of free- and protein-bound 25-OHD in the serum of the patients in this study was not determined, but

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clearly requires to be known. Secondly, it is possible that both dietary vitamin D intake and exposure of the skin to sunlight were decreased in the cirrhotics, with decreased substrate vitamin D available to the liver for 25-hydroxylation. Thirdly, it is possible that cirrhosis may have affected the enterohepatic circulation of 25-OHD (30). In cirrhotics, the enterohepatic circulation of bile acids may be decreased, since deconjugation of cholylglycine as determined by the cholylglycine breath test (31) is decreased (GW Hepner, unpublished observations). H o w e v e r , decreased enterohepatic cycling of 25-OHD would tend to decrease rather than increase its rate of loss from the body, since the rate of exposure to the intestine, the site of loss, would be decreased. Hence altered dynamics of the enterohepatic circulation of 25-OHD in cirrhotics are unlikely to explain the low serum 25-OHD observed in this study. Although chronic alcohol abuse did not cause a significant decrease in serum 25-OHD levels in either the alcoholic controls or the alcoholic cirrhotics, the level of serum 25-OHD in both these groups of patients was lower than in the serum of their nonalcoholic counterparts. It is also of interest that serum 25-OHD in the small group of alcoholic cirrhotics did not correlate with the two indices of hepatic function, albumin and the aminopyrine breath test, in contrast with the correlations found between these parameters in the inactive cirrhotics. It is possible that alcoholism p e r s e may be associated with decreased dietary intake of vitamin D as well as decreased exposure of the skin to sunlight, It is also possible that alcohol may affect vitamin D metabolism, either by decreasing hepatic 25-hyDigestive Diseases, Vol. 21, No. 7 (July 1976)

VITAMIN D METABOLISM IN CIRRHOSIS

droxylation or by increasing catabolism of 25-OHD as has been described after therapy with anticonvulsants such as phenobarbital and diphenylhydantoin (32-34). Such an effect needs to be further investigated. In contrast to patients with chronic renal disease, in whom osteodystrophy is common (9), the bones of cirrhotics show little abnormality apart from osteoporosis (10-12). Since the liver as well as the kidney is required for the conversion of vitamin D to biologically active metabolites, the apparent absence of osteomalacia in cirrhotics is surprising. Further studies on the secretion of parathyroid hormone in these patients are clearly required. ACKNOWLEDGMENTS The vitamin D3 used in this study was a kind gift of Dr. Hector DeLuca. NOTE ADDED IN PROOF After this study was completed, 3 patients with primary biliary cirrhosis were studied. All had normal hepatic microsomal function, as assessed by the aminopyrine breath test. All three were given parenteral injections of vitamin D3 as described for the previous patients in the study. Within 24 hr, serum 25-OHD in the 3 patients rose from 9.3 to 14.6 ng/ml, 17.7 to 30.6 ng/ml, and 20.6 to 38.7 ng/ml. Thus, in the 3 patients with primary biliary cirrhosis, there appeared to be no defective hydroxylation of vitamin D. These data do not support the contention of Wagonfeld et al [Wagonfeld JB, Nemchausky BA, Bolt M, Boyer JL, Rosenberg IH: Low serum 25-hydroxyvitamin D levels (25-OHD) and skeletal demineralisation in patients with primary biliary cirrhosis (PBC): therapeutic response to oral 25-OHD after failure of oral and parenteral vitamin D. Gastroenterology 69:878, 1975 (abstr)] that defective hepatic 25-hydroxylation of vitamin D accounts for the frequent failure of conventional therapy in patients with primary biliary cirrhosis. REFERENCES 1. Bhattacharya MD, DeLuca HF: The regulation of rat liver calciferol. J Biol Chem 248:2964-2973, 1973 2. Bhanacharya MD, DeLuca HF: Comparative studies on the 25-hydroxylation of vitamin D:~ and dihydrotachysterol. J Biol Chem 248:2974-2977, 1973 3. Norman AW, Midgett RJ, Myrtle JF, Nowicki HG: Studies on calciferol metabolism I. Production of vitamin D metabolite 4B from 25-OH-cholecalciferol by kidney homogenates. Biochem Biophys Res Commun 42:1082-1087, 1971 4. Olson EB, DeLuca HF; 25-hydroxycholecalciferol: Direct

Digestive Diseases, Vol. 21, No. 7 (July 1976)

effect on calcium transport. Science 165:405-409, 1969 5. Raisz LG, Trummel CL, Simmons H: Induction of bone resorption in tissue culture: Prolonged response after brief exposure to parathyroid hormone of 25-hydroxycholecalciferol. Endocrinology 90:744-751, 1972 6. Myrtle JF, Haussler MR, Norman AW: Evidence for the biologically active form of cholecalciferol in the intestine. J Biol Chem 245:1190-1196, 1970 7. DeLuca HF: Vitamin D--1973. Am J Med 0:1-12, 1974 8. Kodicek E: The story of vitamin D: From vitamin to hormone. Lancet 1:325-329, 1974 9. Stanbury SW, Lumb GA, Mawer EB: Osteodystrophy devel oping spontaneously in the course of chronic renal failure. Arch Intern Med 124:274-281, 1969 10. Bour H, Banest J-P, Schaison G: Relexions sur les troubles du metabolisme phosphocalcique dans les cirrhoses. A propos d'un cas avec osteomalacie dominante et de l'exploration de huit autres cirrhotiques. Presse Med 71:2772-2776, 1963 11. Collesson L, Grilliat J-P, Mathieu J: L'osteose rarefiante dans les cirrhoses du foie. Presse Med 73:2571-2574, 1965 12. Meunier P, Dechavanne M, Prost G: L'os des cirrhotiques etudie par lecture quantitative de la biopsie osseuse. Lyon Med 229:1089-1095, 1973 13. Saville PD: Changes in bone mass with age and alcoholism. J Bone Joint Surg 47A:492-499, 1965 14. Nilsson BE, Westlin NE: Femur density in alcoholism and after gastrectomy. Calcif Tissue Res 10:167-170, 1972 15. Nilsson BE, Westlin NE: Changes in bone mass in alcoholics. Clin Orthop 90:22%232, 1973 16. Chalmers J, Conacher WDH, Gardner DL, Scott PJ: Osteomalacia--a common disease in elderly women..1 Bone Joint Surg 49B:403-423, 1967 17. DeLuca HF: Vitamin D: a new look at an old vitamin. Nutr Rev 29:179-181, 1971 18. Hepner GW, Vesell ES: Quantitative assessment of hepatic function by breath analysis after oral administration of 14Caminopyrine. Ann Intern Med 83:632-638, 1975 19. Belsey R, Deluca HF, Potts JT: Competitive binding assay for vitamin D and 25-OHD vitamin D. J Clin Endocrinol Metab 33:554-557, 1971 20. Rosen JF, Roginsky M, Nathenson G, Finberg L: 25-hydroxyvitamin D plasma levels in mothers and their premature infants with neonatal hypocalcemia. Am J Dis Child 127:220-223, 1974 21. Atkinson M, Nordin BEC, Sherlock S: Malabsorption and bone disease in prolonged obstructive jaundice. Q J Med 25:29%312, 1956 22. Thompson GW, Lewis B, Booth CC: Absorption of vitamin D:~-:~H in control subjects and patients with intestinal malabsorption. J Clin Invest 45:94-102, 1966 23. Kehayoglou AK, Agnew JE, Holdsworth CD, Whelton MJ, Sherlock S: Bone disease and calcium absorption in primary biliary cirrhosis. Lancet 1:715-718, 1968 24. Dent CE, Stamp TCB: Theoretical renal phosphorus threshold in investigation and treatment of osteomalacia. Lancet 1:857-860, 1970 25. Stamp TCB: Intestinal absorption of 25-hydroxycholecalcifer.ol. Lancet 2:121-123, 1974 26. Avioli LV, Haddad JG: Vitamin D: Current concepts. Metabolism 22:507-531, 1973

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27. Gross JB, Comfort MW, Wollaeger EE, Power MH: Total solids, fat and nitrogen in the feces; study of patients with parenchymatous hepatic disease. Gastroenterology 16:140150, 1950 28. Fast BB, Wolfe S J, Stormont JM, Davidson CS: Fat absorption in alcoholics with cirrhosis. Gastroenterology 37:321324, 1959 29. Rasmussen H, Bordier P: Vitamin D--biochemistry and physiology. The Physiological and Cellular Basis of Metabolic Bone Disease, H Rasmussen, P Bordier (eds). Baltimore, Williams and Wilkins, 1974, pp 207-249 30. Arnaud SB, Goldsmith RS, Lambert P, Go VLW: 25-hydroxyvitamin D:~: Evidence of an enterohepatic circulation

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in man. Proc Soc Exp Biol Med 149:570-572, 1975 31. Hepner GW: Increased sensitivity of the cholylglycine breath test for detecting ileal dysfunction. Gastroenterology 68:8-16, 1975 32. Hahn TJ, Birge SJ, Scharp CR, Avioli LV: Phenobarbitalinduced alterations in vitamin D metabolism. J Clin Invest 51:741-748, 1972 33. Hahn TJ, Hendin BA, Scharp CR, Haddad JG: Effect of chronic anticonvulsant therapy on serum 25-hydroxycalciferol levels in adults. N Engl J Med 287:900-904, 1972 34. Hahn TJ, Hendin BA, Scharp CR, Boisseau VC, Haddad JG: Serum 25-hydroxycalciferol and bone mass in children anticonvulsants. N Engl J Med 292:550-554, 1975

Digestive Diseases, Vol. 2l, No. 7 (July 1976)

Abnormal vitamin D metabolism in patients with cirrhosis.

Abnormal Vitamin D Metabolism in Patients with Cirrhosis GERSHON W. H E P N E R , MD, M A R T I N R O G I N S K Y , MD, and H I N G FAI MOO, PhD To...
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