Blood and Liver Concentrations of Vitamins a and E in Children with Cystic Fibrosis of the Pancreas

Pediat. Res. 6: 26-31 (1972) Cystic fibrosis transport of the pancreas vitamin A liver vitamin E

Blood and Liver Concentrations of Vitamins A and E in Children with Cystic Fibrosis of the Pancreas

BARBARA A. UNDERWOOD1'141 AND CAROLYN R. DKNNING

Institute of Human Nutrition, and Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, New York, USA

Extract The median vitamin A level in plasma of patients with cystic fibrosis of the pancreas (CFP) (18 Mg/100 ml) was less than half that of a control group (39 Mg/100 ml), whereas the median vitamin A level in liver (57 ixg/g) was nearly 3.5 times higher (Table II.). Concentrations of vitamin E in both the plasma and the liver of the patients were less than one-third those of the control group. The data suggest that vitamin A-supplemented children with CFP are unable to maintain normal circulating levels of vitamin A because of a defect in mobilization or transport from storage tissue, rather than because of tissue depletion. Low blood levels of vitamin E, however, reflect tissue depletion and not defective transport.

Speculation The cause of the apparent defect in ability to mobilize or transport vitamin A from the liver in children with CFP may be due to a limited ability to synthesize transport proteins or to a reduced activity of hepatic retinyl ester hydrolase enzymes These alter- natives are under investigation.

Introduction and E usually seen in patients with CFP are a reflec- tion of tissue depletion caused by malabsorption or Lipid absorption is impaired in most children with whether there is a defect in the transport of these cystic fibrosis of the pancreas (CFP). Enzyme replace- vitamins from storage tissues. Levels of cholesterol, ment therapy partially, but inconsistently, corrects the vitamins A and E, and carotene in plasma were deter- problem [1, 8, 19]. Clinical, anatomic, and biochemical mined on blood from 48 randomly selected patients evidences for deficiencies of the fat-soluble vitamins A, with CFP after a 12-18 hr fast. Of these 48, 12 pa- D, E, and K have been described in children with tients died and, at autopsy, plasma and tissue were CFP who received no vitamin supplementation [5, 12, obtained and assayed for vitamins A and E. 22, 23, 24, 27, 28]. Some evidences of deficiency may disappear after feeding the responsible vitamin, but the fasting concentration of vitamin A in plasma often Materials and Methods remains low, and the level of vitamin E responds very All patients were diagnosed as having CFP on the basis slowly, in spite of oral administration of high doses of of the clinical picture, elevated sweat electrolytes, and these vitamins [3, 10, 16]. deficiency of trypsin in the duodenal contents. At the The present study was planned to determine time of diagnosis, all patients were routinely placed on whether the low plasma concentrations of vitamins A daily multivitamin supplements (including at least

26 Vitamin A and E concentrations in cystic fibrosis of the pancreas 27

enzymes, and a high protein, high caloric diet contain- '1'nlle J. Plasma levels of vitamin A, carotene, vitamin E, and ing as much fat as could be tolerated. Supplementa- cholesterol in children with cystic librosis of the pancreas and tion with vitamin E was not routine. in control subjects Blood samples were taken from 48 informed and con- Cyst it l'ibiosis Xoimal senting patients randomly selected from a group of 300 Babies in Baker' A i SI) Median Range hospital who were currently attending the CFP clinic after a X ± si) median fast of 12-18 hr. The subjects studied ranged in age Vitamin A, 29 ± 13 27 7 77 39 ± 14 44 from 6 months to 22 years. Blood was drawn into ,ug/100 ml (48 )2 (47) Carotene, 28 i 19 20 5 -72 153 ± 46 145 Vacutainers containing ethylenediaminetetraacetate Mg/lOOml (48) (47) monohydrate (EDTA). Most of the 18 patients had Vitamin K, 270 i- 155 242 4 586 759 ±171 900 Mg/100 ml (48 1 (4 1 ) two or more specimens analyzed at intervals of 6-12 Cholesterol, 102 ± 23 104 66-146 154 months. mg/100 ml (31 )

Autopsies were performed within 24 hr on the 12 1 Bilker el al. [2] Data based on 642 subjects. patients who died. Liver tissue was removed lrom the - Number in parentheses is the number of subjects studied In most eases ol cystic fibrosis of the pant Teas, the means calculated from two or more speci- central portion of the right lobe and immediately mens analyzed at intervals of 6 12 monlhs were used. frozen in an atmosphere of nitrogen. Skeletal muscle was excised and treated similarly in some patients. of 47 normal children (i-14 years of age admitted to Analyses for vitamins A and E (a-tocopherol) were the hospital for elective surgical procedures, and tor a completed within 1 week of death. group of 642 normal New York City school children Plasma concentrations of vitamin A and carotene 10-13 years of age as reported by Baker et al. [2]. All were determined by the micromethod of Neeld and patients with CFP received daily multivitamin supple- Pearson [21], using trifluoroacetic acid (TEA). Vitamin ments including at least 10,000 IU vitamin A, but no A levels in liver were measured on the nonsaponifiable vitamin E. It was not known whether the hospital extract from 1 g finely homogenized frozen tissue by control group received vitamin supplementation; 29% the TFA method. Pyrogallol was added during saponi- of the New York City school control group were re- fication to minimize oxidative losses. Recoveries of var- ported to receive supplementary vitamins, but no men- iable quantities of vitamin A added to liver tissue were tion was made of the nature of the supplement. consistently above 93%. All parameters measured were considerably reduced Vitamin E levels in plasma were measured by the in the plasma of CFP patients. A significant correla- method of Hashim and Schuttringer [13]. The con- tion was found between plasma levels of vitamins A centration of a-tocopherol in liver was determined spec- and E (r — 0.28, P < 0.05) in the patients but not in trophotometrically after isolation from the nonsaponi- the hospital controls. A significant correlation also was fiable lipids by two-dimensional thin layer chromatog- found between plasma levels of vitamin E and choles- raphy as described by Bieri [4]. Recoveries of 5-20 ^g terol (r = 0.46, P < 0.01). a-tocopherol added to liver tissue were consistently above 90%. Twelve of the forty-eight patients with CFP suc- cumbed to their disease, permitting tissue analyses. Cholesterol was measured by standard procedures in Limited preliminary results on some of those patients an AutoAnalyzer. Total protein in plasma was deter- have been reported [29]. Complete results of vitamin A mined by the biuret procedure; albumin was deter- and albumin levels in the plasma obtained from sam- mined by clectrophoresis on silica gel-coated cellulose- plings before death and at autopsy, and the liver acetate strips. Lipid content of liver was determined weight, lipid concentration, and vitamin A at the time gravimetrically on a chloroform-methanol extract [9]. ol autopsy are presented in Table II. The values for vitamin A and lipid are compared with those of a con- Results trol group of children of similar age, but without CFP, The plasma levels of vitamins A and E and carotene who died suddenly from accidental causes [31]. The from 48 patients with CFP, and cholesterol values lor serum values for the control group were obtained from .SI of those patients, are shown in Table I. The age heart blood taken at autopsy within 21 hr of death. range of this group was (i months-22 years, with a mean The mean vitamin A level in plasma of patients of 11 years. For comparison, data are given for a group 10,000 international units (IU) vitamin A), pancreatic 28 UNDERWOOD AND DENNING

• Studies on plasma and liver in children with cystic hbrosis of the pancreas and in control subjects Plasma Liver

itieilit Sex Age, yr 'I'ime specimen Albumin, Vitamin A, Total wt, Lipid, Vitamin A, death K/'IOO ml Mg/100 ml S/100 g Mg/g

A M 17 13 mos 4.4 77 900 3.8 482 11 F 14 4.5 mos 3.4 23 2.5 mos 2.7 14 1050 8.2 127 C F 8 800 3.7 248 I) F 14 17.5 mos 3.9 20 3 mos 4.5 18 3 wks 3.1 18 850 G.4 68

/•: F ') 0 2.3 10 1000 24.7 23

/•• M 17 23 mos 38 9 mos 4.2 35 0 3.2 G 980 5.6 197 a F 23 22 mos 4.1 29 14 mos 24 9 mos 4.2 26 1 wk 2.9 13 4.5 379 H M 17 15 mos 3.2 22 7 mos 3.3 13 0 1.8 16 4.5 85 i M 11 26 mos 31 12 mos 3.4 18 0 3.0 30 600 7.1 995 j M 9 0 3.7 17 730 5.2 156 A F 12 38 mos 23 4 mos 12 0 2.8 15 700 2.9 230 L F 19 0 2.1 8 4.4 802

Mean i so 3.3 ± 0.7 22 ± 14 5.2 ± 1 .:')> 317 i 3OO2 Median 18 197

Control subjects Mean ± so 3.4 ± 0.53 38 ±113 4.2 i 0.5* 126 ± 104'' Median 39 57

1 Value from patient K is excluded because this patient had extensive biliary cirrhosis. 2 Concentration of vitamin A in liver was significantly higher in the children with CFP than in the control subjecs (P < 0.05). 3 Mean of 9 subjects. 4 Mean of 18 subjects. with CFP was significantly lower, and the mean vi- CFP receiving no supplemental tocopherol were less lamin A level in liver was significantly higher, than than one-third that of a control group [29, 30] (Table the values for the control group. In fact, the median III). Values from patients E and G were omitted be- vitamin A level in plasma of the patients was less than cause a tocopherol supplement had been given before hall.' that of the control group, whereas the median death. The ratio of tissue to plasma vitamin E was vitamin A level in liver was nearly 3.5 times higher. valuable and was not significantly different in the pa- Lipid concentrations in liver were not significantly dif- tients from that in the control group. Vitamin E in the ferent if the value for one patient with extensive liver plasma of the CFP patients correlated with liver «-to- involvement is omitted (patient E). No correlation copherol, both when the latter was expressed on the ba- was found between the vitamin A concentrations in sis of tissue weight (r — 0.6 1, P < 0.05) and on the basis the plasma and the liver of either the subjects wiih of tissue lipid ()• — 0.91, P < 0.01). The correlation CFP or the control subjects. coefficients were higher in the patients than in the Jn contrast to vitamin A, concentrations of vitamin controls. E in both the plasma and the liver of patients with In a few patients who died, the a-tocopherol in skel- Vitamin A and E concentrations in cystic fibrosis of the pancreas 29 etal muscle was measured (Table III). In general, mus- Table III. Vitamin E in plasma and a-tocopherol levels in liver cle contained about the same amount of tocopherol and muscle in unsupplemcnted children with cystic fibrosis of as did the liver. the pancreas and in control subjects Plasma Liver Muscle Patient Xo. of Discussion specimens Vitamin K, or-tocopherol, a-to- Mg/100 ml Mg/g copherol fg/g By repeated measurements over a span of several years on the same patient with CFP, we have found the A 1 370 3.5 3.5 levels of vitamin A in plasma to vary from less than IS 2 172 ± '.) 4.5 5.6 C 6.6 10 fig/100 ml to 20-30 ^g/100 ml (low normal). Vitamin D 2 KiO i 34 4.0 A levels in plasma were seldom found to be above 35 F 3 154 ± 54 3.2 /xg/100 ml, and the fluctuations observed could not II 3 208 ± 76 3.4 2.0 consistently be correlated with abnormal liver func- I 3 226 ± 84 3.2 2.6 tion tests, depressed serum albumin, or the clinical ,1 1 125 3.0 2.4 K 3 123 ± 69 1.0 1.4 condition of the patient at the time of sampling. Sam- I. 1 12 2.6 0.!) ples taken near the time of death or at autopsy, however, usually had lower concentrations of vitamin A Mean 171 ± 96 3.5 ± 1.4 2.9 and albumin than samples obtained earlier in the clin- Median 160 3.2 2.4 ical course of the disease. Vitamin E levels in plasma Control subjects obtained at autopsy had not changed and showed less Mean 708l ± 128 12.82 ± 3.4 fluctuation than vitamin A in serially obtained speci- 1 Mean of 9 subjects. mens. 2 Median levels of vitamin A in plasma of school chil- Mean of 18 subjects. dren in the United States range from 44 to 51 /xg/100 to children with CFP which she attributed to low ml [2, 14, 17, 18], except in depressed areas where a serum lipids. Recently, however, the transport of ret- median of 26 /xg/100 ml is reported [20]. In one study, inol was shown to be dependent upon two carrier only 6.5% of the normal children had vitamin A levels proteins, retinol-binding protein (RBP) and prealbu- in plasma below 30 /j.g/100 ml [18], whereas over 50% min [II, 15], both of which are synthesized in the liver of the children with CFP in our study had values [25]. Most vitamin A is stored in the liver esterified below this figure (Table I). All of the CFP children with long chain fatty acid and presumably is freed received supplemental vitamin A, and one would ex- prior to binding with transport proteins. Our data pect their plasma levels to be above 30 /j.g/100 ml suggest that in children with CFP there is a defect in unless a defect in the absorption or transport of the the ability to mobilize vitamin A from hepatic cells, vitamin existed. perhaps owing to an inability to release retinol or to Lipids are malabsorbed by many CFP patients. The an inability to provide the transport proteins. This low plasma levels of carotene and the low plasma and interpretation is consistent with reports that blood tissue concentrations of vitamin E found in the present levels in CFP patients do rise after vitamin A absorp- study support this fact. The low plasma level of vi- tion tests, but are not maintained at normal levels in tamin A, however, was not solely due to malabsorption spite of prolonged supplementation at high dosages inasmuch as 11 of the 12 patients studied at autopsy [3]. had liver stores of vitamin A which were considerably Our data do not identify the true cause of the de- above the median for a normal population. Only one fect. Like albumin, the vitamin A carrier proteins are patient with extensive cirrhosis and fatty infiltration believed to be synthesized in the liver and liver metab- had a low hepatic concentration. Hence, a sufficient olism is sometimes altered in CFP [6-8]. At autopsy, quantity of vitamin A had been absorbed and depos- the liver from 3 of the 12 patients showed focal biliary ited in the liver to maintain stores even higher than cirrhosis as typically seen in some cases of CFP [7]. those of age-matched controls, and considerably higher The liver of the remaining nine patients showed vary- than those reported for an adult population [31J. ing degrees of chronic passive congestion of the cardiac Normally, stores of vitamin A in liver are exhausted variety and some central necrosis and fibrosis, but before plasma levels decline. In 1939, Andersen [1] there was no evidence of liver disease as gauged by suggested a defect in the transport of vitamin A given liver function tests. Hence, this subclinical liver pa- 30 UNDERWOOD AND DENNING thology noted at autopsy might account for the re- ever, were less than one-third those of the control duced plasma levels of albumin noted in some pa- group. Concentrations of vitamin E in muscle were tients, and possibly of transport proteins. However, also reduced in the seven patients with CFP in whom Strober at al. [26] reported that rates of albumin syn- this tissue was studied. thesis in children with CFP are normal and thai the The data suggest that children with CFP receiving hypoalbuminemia, sometimes seen in older children oral vitamin A supplements are unable to maintain with CFP and in severe pulmonary disease, is pri- normal circulating levels of vitamin A because of a marily due to hemodilution. Hemodilution is not defect in mobilization or transport from storage tissue, likely to be the cause of the low vitamin A levels in rather than because of depleted tissue levels. In con- our studies, inasmuch as no correlation with albumin trast, the low plasma levels of vitamin E reflect tissue levels in serum was found. Also, we cotdd not relate depletion and not defective transport. the blood level of vitamin A with the severity of res- piratory disease as judged clinically. References and Notes In contrast to vitamin A, the content of vitamin E 1. ANDERSEN, 1). H.: Cystic (ibrosis of the pancreas, vitamin A in both plasma and liver was low in the children who dcliciency and bronchiectasis. J. Pediat., 15: 763 (1939). did not receive an a-tocopherol supplement. The ratio 2. BAKER, H., FRANK, O., FEINCOI.D, S., CHRISTAKIS, G., AND ZIFFER, of the levels of u-tocopherol in plasma and tissue was H.: Vitamins, total cholesterol, and triglycerides in 642 New constant, suggesting that both tissues were depleted in York City school children. Amcr. J. Clin. Nutr., 20: 850 (1967). parallel. These low plasma values are similar to those 3. BENNETT, M. J., AND MEDWADOWSKI, B. F.: Vitamin A, vitamin reported by others for children with CFP [3, 12, 16] E, and lipids in serum of children with cystic fibrosis or con- genital heart defects compared with normal children. Amer. and probably reflect malabsorption. Several patients J. Clin. Nutr., 20:415 (1967). received 5 mg/kg/day of a water-dispersible vitamin •1. BII-'RI. J. C: Chroniatography of tocopherols. In: G. V. Mari- E preparation for 4-1 1 months and exhibited an in- netti: Cliromatographic Analysis of I.ipids, Vol. 2, Chapt. 9, crease in plasma values in most cases, but the values p. 159. (Marcel Dckker, New York, 1968). usually remained below normal. Patient (I had re- 5. BI.VNC, W. A., REID, J. D., AND ANDERSEN, D. IL: Avitaminosis ceived this supplement for nearly 1 year belore death F. in cystic fibrosis of the pancreas. A morphologic study of gastrointestinal and striated muscle. Pediatrics. 22: 494 (1958). and piilioit E was given 15 mg/kg/day of u-tocopherol 6. CAKI.N, R., AND CORBO, I..: Plasma fatty acids in pancreatic for 3 months before death. As previously reported [29], cystic fibrosis and liver disease. J. Cliu. F.ndocrinol. Metal)., plasma levels of both these subjects were normal; con- 2(5:470 (1966). centrations in the liver were normal in patient (i and 7. i)i SAN'I' ACNISK, P. A., AND BI.ANC. \V. A.: A distinc:li\e lypc very high in patient E (180 jug/g tissue). of biliary cirrhosis of the liver associated with cystic (ibrosis of the pancreas. Pediatrics, /.V: 387 (1956). (S. DI SANI' ACNESE, P. A., AND I ALAMO, R. C: Pathogenesis and Summary physiopathology of cystic (ibrosis of the pancreas, Iibrocyslii disease of the pancreas (mucoviscidosis). New Engl. J. Mcd., Concentrations of cholesterol, vitamins A and E, and 277: 1399 (1967). carotene were determined in plasma from 48 randomly 9. 1'OI.CH. ).. LEES. M., AND SI.OANE STANI.EV, G. H.: A simple selected patients with CFP. Twelve patients died and, method for the isolation and purification of total lipides from animal tissues. ]. Biol. Ghem., 226: 497 (1957). at autopsy, plasma and tissue were analyzed for vita- 10. COI.DM.OOM, R. B., AND CAMERON, O.: Studies of tocopherol mins A and E. The patients with CFP routinely re- requirements in health and disease. Pediatrics, 32: 36 (1963). ceived supplementary vitamin A (at least 10,000 JU 11. GOODMAN, DE WITT S.: Retinol transport in human plasma. daily), pancreatic enzymes, and a high protein, high Amer. J. Clin. Nutr.. 22: 911 (1969). caloric: diet, but no supplementary vitamin E. 12. GORDON. H. H., NITOWSKY, H. M., AND CORNIU.ATH, M.: Studies of locopherol deficiency in infants and children. Amer. J. All lipids measured were lower in the plasma of His. Child., 9(1: 669 (1955). children with CFP than in that of hospitalized control 13. HASHI.M, S. A., AND Sca-iuriRiNGi K, G. R.: Rapid determination patients of comparable age and in that of normal of locopherol in macro- and microquaniilies of plasma: re- children reported in the literature. sults obtained in a variety of nutritional and metabolic Although the median level of vitamin A in plasma studies in man. Amer. J. Clin. \ulr.. //: 137 (1966). was less than half that of the controls, the median II. HoiK.is, R. E., AND KKIIU., W. A.: Nutritional status of leen- ageis in Iowa. Amer. |. Clin. Nutr., 17: 200 (1965). level in liver for ihe 12 children who came to autopsy 15. KANAI. M., RAZ, A., \ND GOODMAN, HE Win S.: Relinol- was 3.5 times that of the control group. The concen- binding protein: the transport protein for vitamin A in trations of vitamin E in both plasma and liver, how- human plasma. J. Clin. Invest.. -17: 2025 (1968). Vitamin A and E concentrations in cystic fihrosis of the pancreas 31

10. LEVIN, S., GORDON, M. IL, NITOWSKY, IL M., GOLDMAN, C, DI 2(i. STROBER. W., PEIER. (;., AND SCHWARTZ, R. 11.: Albumin SANT' AGNT.SE, P., AND GORDON, H. IL: Studies of tocopherol metabolism in cystic fibrosis. Pediatrics, 13: 116 (1969). deficiency in infants and children. VI. Evaluation of muscle 27. SING, J. II.: Xcuroaxonal dystrophy in mucoviscidosis. J. strength and effect of tocopherol administration in children Xeuropathol. F.xp. \eurol., 23: 507 (1061). with cystic fibrosis. Pediatrics, 27: 578 (1901). 28. TORSI INSON, (). I.., HUMPHREY, G. B.. F.DSON, |. R., AND WAR- 17. MAY, C. I)., BI.ACKEAN, K. 1)., MCCREARY. ). F.. AND ALLEN, WICK, W. J.: Cystic iibrosis presenting with severe hemorrhage F. H., JR.: Clinical studies of vitamin A in infants and due to vitamin K malabsorption: a repon of three cases. children. Amer. J. Dis. Child., 59: 1167 (1040). Pediatrics. 7.5: 857 (1970). 18. MORSE, E. H., MERROW, S. B., AND CLARKE, R. !•'.: Some bio- 29. UNDERWOOD, B. A., AND DENNING, C. R.: Correlations between chemical findings in Burlington (Vt.) junior high school plasma and liver concentrations of vitamins A and E in children. Amer. J. Clin. Nutr., //: 211 (1965). children with cystic fibrosis. Bull. N. V. Acad. Med., 17: 3-1 19. MULI.INCER, M.: The effect of exogenous pancreatic enzymes (1971). on fat absorption. Pediatrics. 12: 523 (1968). 30. UNDERWOOD, B. A., SIEGEL, W. H., DOI.INSKI, M., AND WEISEI.L, 20. MYERS, M. L., MABEL, J. A., AND STARE, F. J.: A nutrition R. C: Liver stoics of a-tocopherol in a normal population study of school children in a depressed urban district. J. dying suddenly and rapidly from unnatural causes in Xew Amer. Diet. Ass., 53: 234 (1968). York City. Amer. J. Clin. Nutr., 23: 1314 (1970). 21. NEELD, J. B., AND PEARSON, W. X.: Macro- and micromethods 31. UNDERWOOD, B. A., SIEGEL, W. H., WEISEI.L, R. C, AND for the determination of serum vitamin A using trifiuoroacetic DOI.INSKI, M.: Liver stores of vitamin A in a normal popu- acid. J. Nutr., 79: 454 (1963). lation dying suddenly or rapidly from unnatural causes in 22. OPPENHEIMER, E. H.: Focal necrosis of striated muscle in an Xew York City. Amer. J. Clin. Nutr., 23: 1037 (1970). infant with cystic fibrosis of the pancreas and evidence of 32. Presented in part at the 40th Annual Meeting of the Socletv lack of absorption of fat-soluble vitamins. Bull. Johns for Pediatric Research, Atlantic City, N. J. May 2, 1970. Hopkins Hosp., 9S: 353 (1956). 33. Supported in part by Whitehall Foundation Research Grant 23. PETERSEN, R. A., PETERSEX, V. S.. AND RORB, R. M.: Vitamin no. 2357. National Institutes of Health Research Grant no. A deficiency with xerophthalmia and night blindness in AM13676, and the National Cystic Fibrosis Research Founda- cystic fibrosis. Amer. J. Dis. Child., lib: 062 (1008). tion. 24. SCHNTIZER. B., AND LoEsi'L, L. S.: Brown bowel. Amer. J. Clin. 31. Requests for reprints should be addressed to: BARBARA A. Path.. 50: 433 (1908). UNDIRUOOD, Ph.D.. Institute of Human Nutrition. College of 25. SMITH, F. R., RAZ, A., AND GOODMAN, DE Win S.: Radio- Physicians and Surgeons. Columbia Universin. 630 W. 168th immunoassay of human plasma retinol-binding protein. J. Street, Xew York, X. Y. 10032 (ISA). Clin. Invest.. 19: 1754 (1970). 35. Accepted for publication April 2, 1971.