Paradoxical Increase of Plasma Vitamin B12 and Folates with Disease Severity in Anorexia Nervosa

EMPIRICAL ARTICLE

Fabiola Corbetta, MD1 ABSTRACT values were found in 55 and 20% of 2 Objective: Anorexia nervosa (AN) is a patients, respectively, a linear correlation Lucio Tremolizzo, MD, PhD complex disorder involving severe psy- with both transaminases was present 2 Elisa Conti, PhD chological manifestations and multiple only for vitamin B12 and folates; further- Carlo Ferrarese, MD, PhD2 organ damage, including liver dysfunc- more, H-B12 patients had both higher 1 tion. The primary aim of this study con- AST and ALT values. EDI- 3 subscores sig- Francesca Neri, PrD sisted in assessing plasma levels of niﬁcantly correlated with vitamin B12 and 1 Monica Bomba, MD vitamin B12 and folates with respect to folates plasma values and H-B12 patients Renata Nacinovich, MD1* liver function enzymes considering the displayed EDI-3 higher values. liver-storage properties of this vitamin. Discussion: These data suggest that

Method: We recruited 70 restrictive plasma levels of vitamin B12 might be an type AN adolescents and the severity early marker of liver dysfunction, possibly of psychopathological traits was also related to more severe psychopatho- assessed using EDI-3 scale. Plasma lev- logical aspects. The identiﬁcation of

els of vitamin B12, folates, transami- patients with higher fasting plasma vita- nases (AST, ALT), gamma-glutamyl min B12 levels could therefore lead to transpeptidase (GGT), alkaline phospha- earlier and more careful refeeding inter- tase (ALP) and cholinesterase (CHE) ventions. Further studies will clarify the were determined. potential role of this vitamin in AN clinical practice. VC 2014 Wiley Periodicals, Inc. Results: About 38.5% of patients displayed vitamin B12 values (H-B12) above Keywords: anorexia nervosa; vita- the upper range of normal reference; min B12; adolescents; liver dysfunc- 4.3% of patients had increased values of tion; transaminases folates; 20 and 11.4% of patients displayed ALT and AST values above refer- (Int J Eat Disord 2015; 48:317–322) ence limits; none had GGT values above normal range. Albeit low CHE and ALP

Introduction with a peak of incidence in adolescence, and is especially alarming given the associated consider- Anorexia nervosa (AN) is a common eating disor- able mortality rate,2 and other medical complica- der (ED) which presents as a complex psychiatric 3,4 tions. Moreover, refeeding syndrome poses condition associated with severe loss of weight and additional risk to AN patients as a relevant meta- excessive concern about body weight and shape bolic disturbance to nutritional rehabilitation.5,6 In with, sometime, weight-regulatory behaviors. The particular, refeeding-associated liver steatosis with prevalence in young females is relatively high,1 increased expression of associated markers is a problem often encountered in clinical practice.7 Accepted 31 October 2014 Interestingly, more than 10% of AN patients show Supported by University of Milano-Bicocca, Italy. disease-related elevation of aminotransferases.5 Fabiola Corbetta and Lucio Tremolizzo contributed equally to the present work. This abnormality is usually asymptomatic and self- *Correspondence to: Renata Nacinovich, MD, University of limiting, but some cases of acute liver failure have Milano Bicocca, Child and Adolescent Mental Health Clinic, S. been described.8–10 The mechanism of liver Gerardo Hospital, via Pergolesi 33, 20900 Monza (MB), Italy. E-mail: [email protected] enzymes elevation observed during severe malnutri- 1 Department of Child and Adolescent Mental Health, San Ger- tion in patients with AN includes hepatocytes ardo Hospital and Department of Surgery and Translational Medi- hypoxia due to low cardiac output and glycogenic cine, University of Milano-Bicocca, Monza, Italy 2 Neurology Unit, San Gerardo Hospital and Department of Sur- depletion, hepatocellular injury from non-alcoholic 11 gery and Translational Medicine, University of Milano-Bicocca, fatty liver disease (NAFLD). oxidative stress, iron Monza, Italy deposition,12 and starvation-induced autophagy.9 Published online 29 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/eat.22371 Body fat and body mass index (BMI) in AN patients VC 2014 Wiley Periodicals, Inc. have been reported as inversely correlated with

International Journal of Eating Disorders 48:3 317–322 2015 317 CORBETTA ET AL. plasma levels of alanine aminotransferase (ALT) and whose role and levels are strictly related to each gamma-glutamyl transpeptidase (GGT), suggesting other) in a sample of 70 adolescent females the presence of NAFLD.11 Consistently, the few liver affected by restrictive type AN, concurrently assess- biopsies available from patients with AN presenting ing liver cytolysis, stasis and synthesis enzymes. As with elevated liver enzymes, show moderate peri- secondary aim, we compared these values with portal inflammatory infiltrates, hepatocyte balloon- clinical and demographic data, including the ing, fatty liver and increased glycogen deposits, assessment of the severity of the psychopathologi- possibly suggesting malnutrition-induced apopto- cal traits by administering the Eating Disorder sis.9 Four specific risk factors for hepatic cytolysis Inventory-3 scale. were identified in a population of adults with AN: age <30 years, BMI <12, male, and the pure restrictive form of the disease.13 Tsukamoto et al.14 Method reported in a cohort of adult ED patients that younger participants presented more pronounced Patient Recruitment metabolic damage deriving from starvation com- Seventy adolescent female inpatients (age between 12 pared with older ones and that lower BMI and and 18 years old) affected by restrictive type AN were shorter disease duration were correlated with ele- diagnosed according to DSM-IV-TR criteria and recruited vated ALT. Montagnese et al.15 further confirmed at the Department of Child and Adolescent Neuropsychia- these findings by identifying a correlation between try of the San Gerardo Hospital, Monza, Italy. Clinical and low BMI and prevalence of hyper-transaminasemia. demographic characteristics are described in Table 1. Moreover, they found an increase in plasma levels Patients with alcohol abuse, cancer (with special attention of lactate dehydrogenase (LDH) and GGT, suggest- to hematological cancers), significant liver or kidney dys- ing generalized liver damage associated with malnu- function, recent infections or surgery were excluded, as trition.15 Low cholinesterase serum levels (CHE, well as patients taking vitamins, steroids or immunosup- marker of hepatocyte function influenced by nutri- pressive drugs, or patients with age <12 and >18 years. tional status) were also found, which inversely cor- Only one patient was taking medications (Sertraline related with hypertransaminasemia.15 50 mg o.d.), while all the others were drug free at the Liver damage is associated not only with hyper- moment of blood withdrawal. Following ethics committee transaminasemia but also with the increase in approval of the study, both parents, and recruited partici- 16,17 pants, gave written informed consent before enrolling in plasma levels of vitamin B12,duetoleakage. the study. Each patient was assessed using the Eating Dis- Normally, the liver stores a vitamin B12 (cobalamin) supply of several milligrams, which is sufficient to order Inventory-3 (EDI-3) in order to have a standardized cover the daily need for several years.18 Diseases that clinical evaluation of symptomatology associated with AN. In the analyses we considered the three eating disor- lead to elevated blood levels of vitamin B12 include hematologic disorders (e.g., acute and chronic der specific scores (DT: drive for thinness; B: bulimia; BD: myelogenous leukaemia), and liver diseases (e.g., body dissatisfaction), the resulting composite (EDRC, eat- acute hepatitis or hepatocellular carcinoma).16–18 ing disorder risk composite), and the composite deriving However, dietary supplementation or integration from the other nine scores not specific for eating disorders (GPMC, general psychological maladjustment composite). that may elevate plasma vitamin B12 should be considered as the primary cause of any increase.18 Disease duration (months), progression rate [defined as The need for predicting liver damage in AN delta BMI (i.e., BMI at disease onset-BMI at current evalu- before full development is strong as it may lead to ation)/disease duration] and BMI were included in the dramatic consequences and influence subsequent analyses as well. weight recovery, especially in younger patients.13 For this reason, the availability of sensitive markers Plasma Determinations of hepatic damage might be crucial for the man- Blood withdrawal for each participant was performed agement of AN in clinical practice. The vitamin B12 on admission or the next day if the admission was during levels of AN patients have never been systemati- the afternoon (in this latter case no drugs were adminis- cally investigated with this aim. This lack of study tered to the patients). Blood was obtained always is due to the general assumptions that, if any differ- between 08.00 and 08.30 AM, following overnight fasting ences were found between the levels of AN patients and immediately sent to the Laboratory of Analyses for and those of the healthy participants, they would plasma separation and the assessment of the variables of be related to the restrictive feeding attitude. Hence, interest.19 Laboratory investigations were carried in this study our primary aim was to assess fasting out blind with respect to clinical data. Fasting plasma plasma levels of vitamin B12 and B9 (or folates, levels of vitamin B12 and folates were assessed by

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TABLE 1. Clinical, demographic, and biochemical characteristics of the enrolled patients Correlation with a N 5 70 AN; mean 6 SD (range) OUT n (%) vitamin B12 Correlation with folates Age (years) 14.7 6 1.6 (12–18) N/A ns ns BMI 15.2 6 1.9 (11.7–19.6) ns ns Disease duration (months) 11.1 6 8.4 (1–38) N/A ns r 5 .3319; p .005701 Progression rate 0.68 6 0.63 (0.03–2.77) N/A ns ns 21 b Vitamin B12 (191–663 pg ml ) 643 6 333 (236–1880) H: 27 (38.5%) ;L:0 — r 5 .4880; p < .0001 Folates (3.1–17.5 ng ml21) 8.4 6 4.5 (2.6–29.5) H: 3 (4.3%); r 5 .4880; p < .0001 — L: 1 (1.4%) AST (<32 U l21) 22.9 6 6.9 (10–40) H: 8 (11.4%) r 5 .3147; p .010705 r 5 .3024; p .014305 ALT (<33 U l21) 22.4 6 13.6 (9–69) H: 14 (20.0%) r 5 .2466; p .039605 r 5 .2791; p .019305 GGT (5–36 U l21) 13.2 6 5.2 (10–32) none ns ns ALP (40–129 U l21) 64.6 6 27.2 (24–132) H: 1 (1.4%); ns ns L: 14 (20.0%) CHE (5,320–12,920 U l21) 5803 6 2179 (3,074–13,565) H: 3 (4.2%); ns ns L: 39 (55.7%) EDI-3 –DT 65.2 6 26.6 (14–99) N/A r 5 .3485; p .004401 r 5 .3127; p .011205 EDI-3 –B 32.7 6 31.9c (0–96) N/A ns ns EDI-3 –BD 57.5 6 28.1 (5–98) N/A r 5 .3189; p .009601 r 5 .3279; p .007701 EDI-3 -EDRC 59.9 6 25.8 (5–94) N/A r 5 .3428; p .005201 r 5 .3144; p .010805 EDI-3 -GPMC 64.7 6 24.0 (8–100) N/A r 5 .3800; p .0018 r 5 .3700; p .002401

In the first column, below each plasmatic biochemical determination normal range values and unit of measure are provided; AN, anorexia nervosa; EDI- 3, Eating Disorder Inventory-3; -DT, drive for thinness; -B, bulimia; -BD, body dissatisfaction; -EDRC, eating disorder risk composite; -GPMC, general psychological maladjustment composite. Correlations are calculated with the two-tailed Pearson’s coefficient and r and p values are provided; N/A, not applicable; ns, not significant; H, high values above normal range; L, low values below normal range. aOUT: number and proportion of cases falling above or below normal-range reference valuesvalues either above upper, or below lower reference limits, here defined according to normal range values (detailed below each parameter) as provided by the Laboratory of Analyses of the San Gerardo Hospital Monza, Italy. bv2 5.83 p < .02 05 vs. ALT (OR 2.51, 95% CI 1.11–5.76) and v2 13.75 p < .0003 001 vs. AST (OR 4.87, 95% CI 1.88–12.95); median and interquartile range: 0.48, 0.03–0.75. cMedian and interquartile range: 29, 0–61. electrochemiluminescence immunoassays. Moreover, range of normal reference ones (191–663 pg mL21), liver cytolysis and stasis were assessed by measuring while none displayed below the lower range (see fasting plasma levels of: aspartate and alanine transami- Table 1). Only 14 and 8 (20.0 and 11.4%) displayed nases (AST and ALT, markers of liver cytolysis), gamma- ALT and AST values above normal reference limits, glutamyl transpeptidase (GGT), alkaline phosphatase respectively (p < .02 and p < .001 vs. vitamin B12, (ALP) (both markers of liver stasis) and pseudocholines- respectively, v2 test, see Table 1). None of the terase (CHE, marker of liver synthesis and hepatocyte patients had GGT values outside the normal range function influenced by nutritional status) on a Cobas ones; additionally, ALP and CHE values were often analyzer by the c 701 module (Roche). The range of nor- lower than normal (20.0 and 55.7% of the cases, mality for each assessed variable was provided by the respectively). Laboratory of Analyses of the San Gerardo Hospital A linear correlation was present between vitamin Monza (Italy). All reference values were verified accord- B12 values and folates or both transaminases (see ing to the procedures of the Clinical and Laboratory Fig. 1 and Table 1). Interestingly, by separating Standards Institute (CLSI; http://clsi.org/). patients according to vitamin B12 upper reference values (above vs. below 663 pg mL21), we found Statistical Analysis that high-B12 (H-B12) patients had both higher AST Two-tailed unpaired Student’s t test was used to assess and ALT values when compared with the other the significance of absolute differences between H-B12 patients (i.e., normal B12 patients, or n-B12) (126% and n-B12 values (see Results). Correlations were com- and 148%, for AST and ALT, respectively, p < .01, puted with the two-tailed Pearson’s r test. v2 test was used two-tailed unpaired Student’s t test; see Fig. 2). for assessing differences of distributions. Statistical analy- Considering only H-B12 patients, no differences sis was performed by Prism 4.00 (GraphPad Software). emerged comparing (see Fig. 1) those expressing normal AST or ALT levels with respect to those with levels above upper reference limits. On the other Results hand, the H-B12 and the n-B12 groups did not significantly differ regarding GGT, ALP, and CHE val- Twenty-seven AN patients (38.5%) displayed fast- ues or other disease characteristics (age, BMI, ing plasma vitamin B12 values above the upper duration, progression rate). Neither ALP, nor CHE

International Journal of Eating Disorders 48:3 317–322 2015 319 CORBETTA ET AL.

FIGURE 1 Correlations between fasting plasma vitamin B12 levels FIGURE 2 H-B12 AN patients (black bars) display higher transami- and transaminases, either AST (A, r 5 .31; p 5 .01, two-tailed Pearson’s r nase levels (both AST and ALT) compared with n-B12 AN patients (white test) or ALT (B, r 5 .24; p < .05). Higher reference limits are indicated by bars). *p < .01 vs. corresponding n-B12 patients, two-tailed unpaired 21 21 21 the lines (vitamin B12: 663.0 pg ml ,AST:32.0Ul ,ALT:33.0Ul ). Student’s t test.

higher just in a minority of patients (4.3%), even if they displayed a moderate-to-strong correlation with vitamin B12 values and a moderate-to-low correlation with transaminases and EDI-3 subscores (see Table 1). Plasma folates analogously correlated with EDI-3 GPMC as well (see Table 1).

Discussion The primary aim of this study was to investigate vitamin B12 level in a sample of adolescent females affected by restrictive type anorexia nervosa (AN), clarifying its putative correlations with markers of correlated with AST and ALT levels, and after com- liver dysfunction, given the liver storage properties paring AN patients with low values of these two of this metabolite. This issue might be of relevance enzymes with patients with normal values no dif- since vitamin B12 values might be usefully marking ferences emerged. Contrarily, GGT values corre- liver dysfunction, possibly guiding clinicians’ lated with both AST (r 5.254; p < .05, two-tailed choices during refeeding of AN patients. Pearson’s r test) and ALT (r 5.419; p < .001), To our knowledge no other studies have system- although they still remained within the normal atically investigated this potential role of vitamin range. B12, and speciﬁcally with reference to AN. Previ- EDI-3 subscores (DT, BD, and EDRC, not B) cor- ously, Zenger et al. described a tendency to an related with vitamin B12 plasma values and were increase in vitamin B12 (and folic acid) plasma lev- lower in n-B12 patients compared with H-B12 ones els in a group of 11 anorexia nervosa adolescents, (all the parameters with -DT just on the border of compared to 12 healthy controls,20 as also shown signiﬁcance, see Fig. 3). The EDI-3 GPMC values by other studies on adolescents and adult 21 correlated with fasting plasma vitamin B12 levels patients. Similarly, we found an increase in fast- (see Table 1) as well, showing higher scores in H- ing plasma vitamin B12; the magnitude of the phe- B12 patients (see Fig. 3). nomenon, however, was quite surprising and, in Folates were higher in the AN H-B12 group com- particular, almost 40% of recruited AN adolescents pared with the n-B12 group (10.8 6 5.8 vs. 7.0 6 2.9 displayed values above the upper range of normal 21 ng ml , 152%, p < .001, two-tailed unpaired Stu- reference. The fact that vitamin B12 levels were not dent’s t test). Nonetheless, folates were overall reduced in nutritionally depleted individuals (AN)

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FIGURE 3 H-B12 AN patients (black bars) display higher scores at reflect liver impairment which can be identified the EDI-3 compared with n-B12 AN patients (white bars). *p < .05 vs. corresponding n-B12 patients; 1p 5 .0580 vs. DT in N-B12 patients, before a significant increase in transaminases. two-tailed unpaired Student’s t test. DT, drive for thinness; B, bulimia; On the other hand, <5% of AN participants pre- BD, body dissatisfaction; EDRC, eating disorder risk composite; GPMC, sented folates values above the normal reference general psychological maladjustment composite. range. Therefore, although folates and vitamin B12 were positively correlated with each other, only vitamin B12 was significantly higher, further indicating that the specific liver storage of this latter vitamin plays a role in generating the difference. In our AN population, ALP and CHE values were also below the normal range in considerable percentages of patients, as shown by other studies, too.15,22 Neither CHE, nor ALP,however, were correlated with transaminases and comparing those patients with low levels of the two enzymes compared with patients displaying normal levels, no differences emerged relative to AST, ALT, and vitamin B12 levels. In this study we also made a comparison between biochemical and clinical data and we did not find any correlation between vitamin B12 plasma levels and BMI, progression rate and disease duration. This is in contrast with the hypothe- certainly deserves further reflections. The finding sis of marking disease severity by using vitamin could not have been due to dietary supplementation B12. However, we found positive correlations 18 or integration, and was most probably due to the between vitamin B12 and EDI-3 eating disorders presence of liver dysfunction, inducing B12 hypervi- specific subscales and general composites. Fur- 16,17 taminosis by leakage, as already suggested. The thermore, H-B12 patients obtained overall higher detection of an increase in ALT and AST values, EDI-3 scores than n-B12 ones. We could hypothe- above normal upper limits, respectively, in 20 and size that these patients might present worse symp- 12% of the participants, allowed us to confirm the tomatic conducts (e.g., severe food and water presence of moderate liver dysfunction (maximum intake restriction). BMI and EDI-3, in fact, assess 21 values were 40.0 and 69.0 U l for AST and ALT, AN severity according to completely different respectively, see Table 1) in our population, in approaches and the possibility that factors other agreement with several previous reports on this dis- than malnutrition might contribute to determine 5,6,10 ease. Interestingly, apart from the elevation of liver damage can be raised.23 Additionally, folates liver markers, our patients did not display clinical correlate with transaminases and EDI-3 scores. symptoms of liver failure. We therefore hypothesize They also correlate with disease duration, possibly that vitamin B12 might be marking preclinical liver suggesting that putative dysfunctions might dysfunction more sensibly than transaminases. Fur- become operative after time, offering potential thermore, one limit might be indicated in the fact opportunities for early interventions. The alteration that we did not specifically exclude chronic hepati- of blood vitamin B12 levels could lead to the tis, albeit the age group and the lack of significantly impairment of its role of coenzyme assisting the elevated liver function tests minimizes this possibil- synthesis of purines and pyrimidines and the deg- ity. Furthermore, history of major liver disorders, radation of fatty acids. Moreover, vitamin B12,in including hepatitis, and cancer were all exclusion association with acid folic and homocysteine, is criteria in our study, with the implicit limit of involved in the one-carbon cycle that governs DNA obtaining this information from the history and not methylation. This process is altered in AN patients from specific tests. that display reduced global DNA methylation in 24–26 A correlation between AST/ALT and vitamin B12 whole blood samples. Finally, circulating vita- levels was present, and by dividing patients into two min B12 has two binding proteins, i.e., trans- 27 groups based on vitamin B12 values we showed that cobalamin and haptocorrin. One limitation of the hypervitaminosis patients (H-B12) had higher trans- present study consists in the lack of data regarding aminases levels than normovitaminosis patients (n- these binding proteins, since reduced liver synthe- B12). These data might be reasonably interpreted sis might eventually lead to reduced circulating hypothesizing that high cobalamin plasma levels levels.27

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