SERUM FERRITIN AND IRON LEVELS IN CHRONIC MALE ALCOHOLICS BEFORE AND AFTER ETHANOL WITHDRAWAL

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1 Atcohotd Alcoholism, Vol. 18, No. 3, pp , 1983 rinted in Great Britain O3O9-1635/83S3.OO ergamon ress Ltd Medical Council on Alcoholism SERUM FERRITIN AND IRON LEVELS IN CHRONIC MALE ALCOHOLICS BEFORE AND AFTER ETHANOL WITHDRAWAL MATTI VALIMAKI*, MATTI HARKONEN and REINO YLIKAHRI Third Department of Medicine and Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland and Research Laboratories of the State Alcohol Monopoly (Alko), Helsinki, Finland (Received 17 January 1983) Abstract Serum ferritin and iron concentrations were determined before and during 2 weeks of alcohol abstinence in 14 chronic male alcoholics with and in 12 alcoholic men without these signs. As compared to the controls, the serum ferritin level was elevated in both groups immediately after a drinking bout, significantly more so in men with, than in those without, of liver injury. The serum ferritin was normalized after one and two weeks of alcohol abstinence in alcoholics without and with of liver disease, respectively. The serum iron concentration was equally increased, but returned to normal during the first week of ethanol withdrawal in both groups. No correlations were found between the absolute serum ferritin and iron values nor between changes therein. The values for serum ferritin, unlike those for the serum iron, correlated positively with the degree. Acute ethanol intake was without effect on the serum ferritin and iron concentrations in normal healthy controls. Our results demonstrate that in alcoholics liver injury contributes to the increase in the serum ferritin, but not to that in the serum iron. INTRODUCTION An increase in serum concentrations of iron is often seen in people consuming large amounts of ethanol (Sullivan and Herbert, 1964; Waters et al., 1966; Lindenbaum and Lieber, 1969; Hines and Cowan, 1970; Hourihane and Weir, 1970; Eichner and Hillman, 1971; Myrhed et al., 1977; Lundin et al, 1981). This may result from increased iron ingestion in some alcoholic beverages, from increased iron absorption (Charlton et al., 1964) especially in connection with folic acid deficiency (Celada et al., 1979) and from an ethanol-induced block in the normal utilization of iron (Sullivan and Herbert, 1964; Waters et al., 1966; Hines and Cowan, 1970, Eichner and Hillman, 1971). Apart from high levels of serum iron, chronic alcoholics often have increased serum concentrations of ferritin (Lundin et al., 1981). Ferritin is an iron storage protein, present mainly in the cells of the liver and reticulo-endothelial system. The serum concentration of this protein usually reflects the magnitude of the iron stores Correspondence should be addressed to Dr Matti Valimaki, Third Department of Medicine, University Central Hospital, SF Helsinki 29, Finland. in the body (Lipschitz et al., 1974). In alcoholics with liver damage the usefulness of this test for evaluation of storage iron is, however, limited, since the concomitant liver injury elevates the serum ferritin level independently of any change in the iron stores (rieto et al., 1975). Thus, in spite of high serum levels of ferritin, iron stores have been shown not to be increased in chronic alcoholics (Lundvall et al., 1969). In the present study we have attempted to clarify whether the increases in serum ferritin and iron in chronic alcoholics are dependent on each other and whether they derive their origins from the same source. With this aim we studied the serum iron and ferritin concentrations in two groups of alcoholics differing from each other with regard to the presence of. The effect of acute ethanol intake on the same parameters of iron metabolism has also been examined in normal healthy controls. ATIENTS AND METHODS The study, which had been approved by the University Ethical Committee, was carried out 255

2 256 M. VALIMAKI, M. HARKONEN and R. YLIKAHRI on 26 male alcoholics (aged years old) who volunteered for withdrawal therapy at a special institution for the treatment of alcoholics (Kylasaari Asylum, Helsinki). The purpose and design of the study was explained to each subject and formal consent was obtained. After the patients had agreed to participate in this study, they were transferred to the University Hospital. Each subject was still intoxicated on admission. The duration of their drinking history varied from 10 to 25 years and that of the last continuous period of daily drinking from 2 weeks to 2 years. Apart from their alcoholism, the patients were otherwise healthy and were not taking any drugs. The stay at the University Hospital lasted for 1 to 2 weeks during which the patients received an isocaloric diet (30 kcal/kg body wt per day) with approximately 45% of the total energy as carbohydrate, 35% as fat and 20% as protein. No alcohol was allowed. The investigations were carried out on the day following admission and were repeated after 1 and 2 weeks of abstinence from alcohol. On the morning of each study day (after a 12-hr fast), a venousblood sample was taken for the determination of serum iron, transferrin and ferritin. The first sample was also used to estimate the following parameters: serum gamma-glutamyltranspeptidase (y-gt), aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activities and basic haematological parameters. The patients were divided into two groups on the basis of the serum y-gt value. Twelve men having a normal serum y-gt formed the group without, and 14 alcoholics with an abnormal serum y-gt formed the group with liver damage. Eleven non-alcoholic healthy persons (aged years) who had abstained from consuming ethanol for at least one week, served as controls. These same volunteers also participated in a study in which they consumed ethanol (5.5 g/kg body wt) as a 15% solution in water and lingonberry juice during the 54 hr and the above mentioned diet. The parameters of iron status were determined before and after the drinking bout. Serum ferritin concentration was measured using hadebas Ferritin rist radioimmunoassay kit (harmacia Diagnostics, Uppsala, Sweden). In every series of determinations, control serum was included and a quality control chart was plotted. The imprecision between assays (at the level of 20 ng/1) was 10.3% and the reference values for men were ng/1. The serum iron, transferrin, y-gt, ASAT and ALAT as well as blood haemoglobin concentration and erythrocyte parameters were determined with methods routinely used in clinical laboratories (Committee on Enzymes; the Scandinavian Society for Clinical Chemistry and Clinical hysiology, 1974, 1976). In the statistical analysis both the paired and unpaired forms of Student's Mest were used. Linear regression analysis was used for calculation of correlation coefficients between the parameters measured. RESULTS Clinical data and basic laboratory parameters Clinical data and values for basic laboratory parameters in the study groups are presented in Table 1. The alcoholics with were somewhat older than the patients without these signs, but the duration of alcohol abuse and that of the last drinking bout were not different between the study groups. Blood haemoglobin values were normal in both groups. Of the corpuscular values for the erythrocytes the only difference between the groups was found in the mean corpuscular haemoglobin concentration, which was somewhat lower in the alcoholics with signs of liver injury than in the patients without these signs. In addition to serum y-gt, the groups also differed significantly from each other in the values for serum ASAT and serum ALAT (Table 1). Serum ferritin On admission the alcoholics with liver damage had higher serum concentrations of ferritin ( ug/1; mean SEM) than the alcoholics without signs of liver injury (138 15; < 0.001) or the control men (66 15; < 0.001) (Fig. 1). The difference between the latter groups was also statistically significant ( < 0.01). Of 14 alcoholics with liver disease the serum ferritin values were above our normal range in 10, whereas of 12 alcoholics without

3 SERUM FERRITIN AND IRON LEVELS IN ALCOHOLICS 257 Table 1. Clinical status of the patients. Clinical data and values for basic laboratory parameters in the study groups Alcoholics with biochemical signs of liver damagf Alcoholics without biochemical signs of liver damage Reference values Number of patients Age (years) Duration of alcohol abuse (years) Duration of last drinking bout (months) Blood haemoglobin (g/1) Blood erythrocytes (10 l2 /l) Mean corpuscular haemoglobin (pg/cell) Mean corpuscular haemoglobin concentration (g/1) Mean corpuscular volume (fl) Serum gamma-glutamyltranspeptidase (U/l) Serum aspartate aminotransferase (U/l) Serum alanine aminotransferase (U/l) [ 1.2* * *** 47.2*** 12.0*** < 50 < 40 < 40 All values are means SEM. * < 0.05, *** < for differences between the groups. of hepatic damage, only one showed an abnormally high value. During the first week of alcohol abstinence the serum ferritin concentration was reduced by 49% and 56% in the groups with and without signs of liver damage, respectively ( < 0.001) (Fig. 1). During the second week of abstinence serum ferritin was further decreased in the alcoholics with liver damage (Fig. 1). Serum iron and transferrin The initial values for the serum iron concentrations were similar in both study-groups (alcoholics with liver damage ixmol/l and alcoholics without liver damage ) and differed significantly from those found in the controls ( ; < 0.001) (Fig. 2). Abstinence from ethanol for one week resulted in decreases of 61 % and 57% ( < 0.001) respectively for the patient groups (Fig. 2). During the second week of alcohol abstinence the serum iron level fell further in both groups, significantly so, however, only in the patients with liver disease ( < 0.05). Immediately after a drinking bout the serum transferrin level did not differ significantly between the alcoholics with liver injury ( g/1) and those without of hepatic damage ( ). During the first week of abstinence the transferrin level rose to 500 r i I 300 c I 200 "E Duration of abstinence ( wk ) Fig. 1. Serum ferritin during alcohol abstinence. Serum ferritin concentrations in normal healthy controls (A) and in abstaining male alcoholics with ( ) and without (O). *** < and ** < 0.01 for the difference between the study-groups and for that from values of controls, t>o < 0.01 for the difference from values of controls. Results are means SEM.

4 258 M. VAL1MAK1, M. HARKONEN and R. YL1KAHRI I c o E I Duration of abstinence ( wk ) Fig. 2. Serum iron concentrations during alcohol abstinence. Serum iron concentrations in normal healthy controls (A) and in abstaining male alcoholics with(»)and without (O). <rft» < for the differences from values of controls. Results are means SEM in the former group ( < 0.05), but remained unchanged in the latter. Correlations between the parameters of iron status The correlations between the serum ferritin and iron values are presented in Table 2. Immediately after a drinking bout, no correlations were found between these two parameters. One week after stopping drinking, a slight correlation (r = 0.544, < 0.05) was observed in the alcoholics with liver damage. After alcohol abstinence for two weeks the serum ferritin and iron values correlated positively in the alcoholics without signs of hepatopathy (r = 0.859, < 0.05). There were no correlations between the changes measured in the serum ferritin and iron during the first and second week of follow-up, respectively. An inverse correlation was found immediately after a drinking bout between the serum ferritin and transferrin values in the alcoholics with hepatopathy (r = , < 0.05) and in the combined group of alcoholics (r = , < 0.05). These correlations disappeared after alcohol abstinence for one week. Correlations between the parameters of liver damage and those of iron status During the early phase of alcohol abstinence, serum ferritin concentration correlated positively with activities of both serum y-gt (r = 0.761, < 0.001) and serum ASAT (r = 0.681, < 0.001) in the combined group of alcoholics (Table 3), whereas a less strong correlation was found between serum ferritin and serum ALAT activity (Table 3). In the patients with liver damage, serum ferritin concentration correlated positively with activities of serum Y-GT (r = 0.678, < 0.01), serum ASAT (r = 0.670, < 0.01) and serum ALAT (r = 0.600, < 0.05) (Table 3). No correlations were found between serum iron concentrations and the parameters of liver function. Iron status after acute alcohol intake Acute alcohol ingestion by normal healthy controls was without effect on both the serum concentration of ferritin (before: Hg/1; after: ) and that of iron (before: umol/1; after ). Table 2. Relations between serum ferritin and iron concentrations. The correlations between the serum ferritin and iron values in the abstinent alcoholics. Correlation coefficients and their significance as well as number of patients are expressed Duration of abstinence (wk) Whole group Alcoholics with Alcoholics without N r N r N < < 0.05 = not significant

5 SERUM FERRITIN AND IRON LEVELS IN ALCOHOLICS 259 Table 3. Relations of serum ferritin to liver damage. The correlations between the serum ferritin and the parameters at the beginning of alcohol abstinence. Correlation coefficients and their significance are expressed Assay Whole group (N = 26) Alcoholics with (N = 14) Alcoholics without (N = 12) r r r y-gt ASAT ALAT < < < < < < = not significant DISCUSSION The present study clearly shows that although in abstinent alcoholics the serum concentrations of ferritin and iron change in a parallel fashion, they are not related to each other, even in alcoholics without biochemical signs. Thus, our results exclude the possibility that the increment in iron in the serum is due to the increased ferritin. This possibility is made unlikely also by the fact that in liver diseases iron content of ferritin is very low (Zuyderhoudt et al., 1978). Furthermore, confirming the findings of Lundin and his coworkers (1981), our study shows that in chronic alcoholics the elevations of the serum ferritin and iron levels derive their origins from different causes. The pronounced difference in the serum ferritin between our study-groups and the correlations found between the serum ferritin and the parameters of hepatic deterioration support the earlier suggestion (rieto et al., 1975; Lundin et al., 1981) that the increased serum ferritin concentrations in alcoholics result mainly from alcohol-induced liver damage. Furthermore, although the serum y-gt activity is a sensitive indicator of ethanol-induced liver injury, it is quite possible that slight hepatic damage is not reflected in the serum level. Thus, liver injury still may be the reason for the increased serum concentration of ferritin in the patients without. In experimental animals acute hepatocellular necrosis can also cause elevation of the serum iron level (Reissmann et al., 1954). However, in our patients, the increased serum iron does not seem to be due to liver damage, because we could not find any correlation between the serum iron and the parameters. Furthermore, serum iron concentration was equally increased in both our study-groups, independently of the state of the liver. As compared to acute hepatocellular necrosis induced under experimental conditions, liver damage associated with alcohol abuse is more chronic, which may in part explain the differences between animal and human studies. There is much experimental evidence supporting the suggestion that the ethanol-induced impairment of iron utilization by red cells is the major mechanism leading to the increased serum concentrations of iron in alcoholics. In ferrokinetic studies, the increase in the serum iron concentration has been shown to occur concomitantly with reduced erythrocyte iron utilization induced by ethanol (Waters et al., 1966; Hines and Cowan, 1970). It was demonstrated recently that in tissue culture, colony formation by erythroid progenitor cells was suppressed by concentrations of ethanol that are easily achieved in vivo (Meagher et al., 1982). Apart from direct interference with erythropoiesis, ethanol may also impair iron utilization by red cells via deficiencies and deranged metabolism of folate and pyridoxine (Sullivan and Herbert, 1964; Hines and Cowan, 1970; Eichner and Hillman, 1971). In patients with anaemia from different causes, as well as in patients with disorders of

6 260 M. VALIMAKI, M. HARKONEN and R. YLIKAHRI iron metabolism, a reciprocal correlation has been found between the serum ferritin and the serum total iron-binding capacity (Lipschitz et al., 1974). In agreement with this observation, we found an inverse correlation between the serum ferritin and transferrin values in our patients immediately after they stopped drinking. It has been suggested that the serum ferritin can be used as an indicator of alcohol consumption in populations (Kristenson et al., 1981). However, compared to the sensitivity of serum y-gt as a measure of alcohol consumption, serum ferritin determination does not seem to offer any advantages. In an earlier report, an elevated serum ferritin level was found in 67% of heavy drinkers with an increased serum y-gt, whereas in individuals with strictly normal serum y-gt only one showed a high serum ferritin level (Kristenson et al., 1981). Similarly, in our study the serum ferritin level was elevated in 71% of the alcoholics with abnormally high serum y-gt, whereas only one patient showed an increased serum concentration of ferritin associated with a normal serum y-gt. By contrast, in all our patients with normal serum y-gt a pronounced fall (varying from 37% to 83%) in serum ferritin concentration was seen during the first week of alcohol abstinence. Consequently, the decrease observed in a patient's serum ferritin during follow-up could be a better indicator of alcohol consumption than the result of one sporadic determination of serum ferritin, perhaps also a better one than serum y-gt activity. Our results show that in alcoholics the serum ferritin and iron levels rise independently of each other and that the increase in the serum ferritin, but not that in the serum iron, is of hepatic origin. Acknowledgements We are grateful to Dr aivi Karkkainen, Kylasaari Asylum, Helsinki, for allowing her patients to be studied. We also thank Miss Leena Tanner for skilled technical assistance. REFERENCES Celada, A., Rudolf, H. and Donath, A. (1979) Effect of experimental chronic alcoholic ingestion and folic acid deficiency on iron absorption. Blood 54, Charlton, R. W., Jacobs,., Seftel, H. and Bothwell, T. H. (1964) Effect of alcohol on iron absorption. British Medical Journal 2, Committee on Enzymes; the Scandinavian Society for Clinical Chemistry and Clinical hysiology (1974) Recommended methods for the determination of four enzymes in blood. Scandinavian Journal of Clinical and Laboratory Investigation 33, Committee on Enzymes; the Scandinavian Society for Clinical Chemistry and Clinical hysiology (1976) Recommended methods for the determination of Y-glutamyltransferase in blood. Scandinavian Journal of Clinical and Laboratory Investigation 36, Eichner, E. R. and Hillman, R. S. (1971) The evolution of anemia in alcoholic patients. American Journal of Medicine 50, Hines, J. D. and Cowan, D. H. (1970) Studies on the pathogenesis of alcohol-induced sideroblastic bone-marrow abnormalities. New England Journal of Medicine 283, Hourihane, D. O'B. and Weir, D. G. (1970) Suppression of erythropoiesis by alcohol. British Medical Journal 1, Kristenson, H., Fex, G. and Trell, E. (1981) Serum ferritin, gammaglutamyltransferase and alcohol consumption in healthy middle-aged men. Drug and Alcohol Dependence 8, Lindenbaum, J. and Lieber, C. S. (1969) Hematologic effects of alcohol in man in the absence of nutritional deficiency. New England Journal of Medicine 281, Lipschitz, D. A., Cook, J. D. and Finch, C. A. (1974) A clinical evaluation of serum ferritin as an index of iron stores. New England Journal of Medicine 290, Lundin, L., Hallgren, R., BirgegSrd, G. and Wide, L. (1981) Serum ferritin in alcoholics and the relation to liver damage, iron state and erythropoietic activity. Ada Medica Scandinavica 209, Lundvall, O., Weinfeld, A. and Lundin,. (1969) Iron stores in alcohol abusers. 1. Liver iron. Ada Medica Scandinavica 185, Meagher, R. C, Sieber, F. and Spivak, J. L. (1982) Suppression of hematopoietic-progenitor-cell proliferation by ethanol and acetaldehyde. New England Journal of Medicine 307, Myrhed, M., Berglund, L. and Bottiger, L. E. (1977) Alcohol consumption and hematology. Ada Medica Scandinavica 202, rieto, J., Barry, M. and Sherlock, S. (1975) Serum ferritin in patients with iron overload and with acute and chronic liver diseases. Gastroenterology 68, Reissman, K. R., Boley, J., Christianson, J. F. and Delp, M. H. (1954) The serum iron in experimental hepatocellular necrosis. Journal of Laboratory and Clinical Medicine 43, Sullivan, L. W. and Herbert, V. (1964) Suppression of hematopoiesis by ethanol. Journal of Clinical Investigation 43, Waters, A. H., Morley, A. A. and Rankin, J. G. (1966) Effect of alcohol on haemopoiesis. British Medical Journal 1, Zuyderhoudt, F. M. J., Linthorst, C. and Hengeveld,. (1978) On the iron content of human serum ferritin, especially in acute viral hepatitis and iron overload. Clinica Chimica Ada 90,