Postmortem Diagnosis of Anorexia Nervosa : An Endocrinological and Immunohistochemical Approach

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1 305 CASE REPORT Postmortem Dignosis of Anorexi Nervos : An Endocrinologicl nd Immunohistochemicl Approch Akiko Ishigmi 1, Itsuo Tokung 1, Akiyoshi Nishimur 1, nd Shin-ichi Kuo 2 1 Deprtment of Forensic Medicine, Institute of Biomedicl Sciences, Tokushim University Grdute School, Tokushim, Jpn, 2 Deprtment of Forensic Medicine, Fculty of Medicine, Fukuok University, Fukuok, Jpn Astrct : A femle in her 30s ws found ded fter fire. She ws severely emcited nd hd een dignosed with norexi nervos (AN) out 5 yers go, ut hd not een treted recently. Therefore, we investigted not only her cuse of deth ut lso her condition of AN. Some of her orgns weighed less thn norml lthough no cler lesions were oserved. In the pituitry glnd, the numer of follicle-stimulting hormone-immunopositive cells ws mrkedly decresed lthough norml numer of thyroid-stimulting hormone-positive cells were detected. A histologicl exmintion of the ovry suggested tht she hd een suffering from menorrhe. The thyroid glnd ws trophic, nd mrked vritions in folliclesizewereoserved.becusewecouldnototin enough volume of her lood for endocrinologicl exmintions, we tried to investigte her endocrinologicl condition y immunohistochemistry. Immunohistochemicl stining detected decresed triiodothyronine immunorectivity nd norml thyroxine immunorectivity. The drenl glnds were lso trophic. Bsed on these findings, it ws considered tht she hd een suffering from AN t the time of her deth. The utopsy nd other findings reveled tht she hd died of urning with cron monoxide intoxiction. J. Med. Invest. 63 : , August, 2016 Keywords : norexi nervos, endocrine disturnce, immunohistochemistry, postmortem dignosis, forensic pthology 1. INTRODUCTION Anorexi nervos (AN) is the one of min types of eting disorder. The Dignostic nd Sttisticl Mnul of Mentl Disorders, Fifth Edition, (DSM- V) defined the dignostic criteri for AN s follows : 1) energy intke restriction reltive to the pproprite nutritionl requirements, leding to significntly low ody weight in the context of ge, sex, developmentl trjectory, nd physicl helth ; 2) n intense fer of gining weight or ecoming ft, even t significntly low weight ; 3) disturnce in the wy in which one s ody weight or shpe is experienced, n undue influence of ody weight or shpe on self-evlution, or persistent lck of recognition of the seriousness of one s current low ody weight (1). Here, we report forensic utopsy cse of AN, in which ody ws found fter fire, together with n investigtion of the pthologicl findings. The im of this study ws to confirm tht the suject ws suffering from AN t the time of her deth. 2. CASE REPORT A fire roke out house erly in the morning in Decemer, nd femle in her 30s ws found ded fter the fire. Medicl history : She hd een emotionlly unstle for 7 yers fter delivering her first child nd hd een dignosed with AN 5 yers go. However, she hd not een treted for AN within the pst 6 months nd hd refused to consume nything except yogurt nd liquids. Received for puliction My 9, 2016 ; ccepted June 16, Address correspondence nd reprint requests to Akiko Ishigmi, Deprtment of Forensic Medicine, Institute of Biomedicl Sciences, Tokushim University Grdute School, , Kurmoto-cho, Tokushim , Jpn nd Fx : Autopsy findings The suject ws severely emcited (height : 158 cm ; weight : 27 kg, ody mss index : 10.8), nd her hed nd fce were covered in urns. Her left lims nd the left side of her trunk were lso urnt (Fig. 1). Aprt from the urn injuries, no other ovious wounds were oserved on her ody. There ws lrge mount of soot in her trche nd ronchus (Fig. 1). A smll mount of soot ws lso found in the esophgus nd stomch. No therml dmge ws seen in the lryngophrynx or glottis. Her hert ws significntly smll, weight ; 126 g,rin nd other orgns hd no ovious lesions. However, vrious orgns weighed less thn norml. In prticulr, the drenl glnds, uterus, nd ovries hd shrunk mrkedly (Tle 1) (2). Her stomch ws empty, nd smll mount of gry-yellow sustnce ws found in the intestines. Toxicologicl exmintions : The sturtion level of cron monoxide (CO)- hemogloin in her left crdic lood ws 82%. No other drugs or sustnces such s ethnol were detected. Endocrinologicl exmintions : Unfortuntely, we could not otin enough volume of her lood for exmintion. 2.2 Histopthologicl findings Tissue smples were fixed with phosphte-uffered formlin, emedded in prffin, nd sectioned t 5 μm. The sections were stined with conventionl stins such s hemtoxylin-eosin (HE). The myocrdil fiers in her hert hd thinned (Fig. 2). No mrked reduction in cell numer or chnges in cell structure were seen in the pituitry glnd (Fig. 3). A histologicl exmintion of the ovries did not revel ny vesiculr ovrin follicles or second ovrin follicles. A corpus luteum nd few primry ovrin follicles were oserved. Mrked vritions in follicle size were oserved in the thyroid (Fig. 4). The drenl glnd seemed to e morphologiclly norml. No significnt chnges were seen in her rin. The lungs, spleen, liver, nd kidneys were congested. The Journl of Medicl Investigtion Vol

2 306 A. Ishigmi, et l. Postmortem Dignosis of Anorexi Nervos ; outer view of the urn injuries, ; the soot in the suject sirwy Fig. 1 Mcroscopic findings of the cdver : outer view of the urn injuries, : the soot in her irwy Tle 1 The weights of the suject s orgns (comprison with the men weights of norml Jpnese orgns) Orgn Weight Men SD Orgn Weight Men SD Hert Pituitry glnd Lung Thyroid left Adrenl glnd right left Kidney right left Uterus right with dnex Liver Spleen Pncres Thymus ; this cse. ; 38 y.o., femle. The cuse of deth ws drowning. The myocrdil fiers hd thinned in this cse. Fig. 2 Microscopic findings of the left ventricle (HE stining) The myocrdil fiers hd thinned. 2.3 Immunohistochemicl findings Immunostining ws performed with ntiodies ginst folliclestimulting hormone (FSH, 1 : 50, Cell Mrque, USA), thyroidstimulting hormone (TSH, 1 : 200, Cell Mrque, USA), triiodothyronine (T3, 1 : 200, Novus Biologicls, USA), thyroxin (T4, 1 : 200, Novus Biologicls, USA), nd cortisol (1 : 100, Chemicon, USA). The immunostining ws crried out using the VECTASTAIN universl quick kit (Vector Lortories, USA) ccording to the mnufcturer s instructions. The immunostining ws visulized y incuting the tissue slices with 0.02% 3,3 - diminoenzidine (DAB) nd 0.03% hydrogen peroxide in Tris-uffered sline. A coverslip ws plced on ech glss slide, nd then the slides were exmined nd photogrphed. Negtive controls, which were used to determine the specificity of the immunohistochemicl rections, were performed y omitting the primry ntiody. The immunohistochemicl findings were exmined seprtely y two pthologists nd were then screened. An immunohistochemicl study of the pituitry glnd detected decresed numer of FSH-positive cells, ut norml levels of TSH immunorectivity (Fig. 3c nd 3e). In the immunohistochemicl exmintion of thyroid hormone expression, T3 immunorectivity ws decresed nd immunorectivity for T4 ws stronger thn T3 (Fig. 4c nd 4e). Norml levels of cortisol immunorectivity were oserved.

3 The Journl of Medicl Investigtion Vol. 63 August this cse control HE stining nti-fsh c d nti-tsh e f, c, e ; this cse., d, f ; 19 y.o., femle. The cuse of deth ws drowning. A numer of FSH-positive cells were decresed, ut norml levels of TSH immunorectivity. Fig. 3 Immunohistochemicl findings of the pituitry glnd, : HE stining, c, d : nti-fsh stining, e, f : nti-tsh immunostining 3. DISCUSSION AN is n eting disorder nd hs prevlence of out 0.048% in Jpnese women (3). Ptients with AN exhiit vrious physicl findings nd psychitric disorders s complictions (Tle 2) (1, 4, 5). In the present cse, lmost ll of her orgns hd trophied nd were light. Thus, the suject seemed to hve een severely mlnourished. Her hert weighed less thn hlf the weight of norml hert nd contined little dipose tissue. In ddition, the myocrdil fiers hd thinned nd trophied. Thus, it ws suspected tht her hert hd filed. Endocrine nd metolic disturnces re known to occur in AN (5, 6). Severe cloric restriction suppresses the hypothlmicpituitry xis, nd ptients with AN exhiit hypogondotropichypogondism of hypothlmic origin (7). Clinicl dignosis of AN is prtly sed on endocrinologicl exmintions. However, in our forensic utopsy cse, we could not otin enough lood for those exmintion. So, we tried to investigte her endocrinologicl condition y immunohistochemistry. Histologicl exmintion did not detect ny ovious normlities in the pituitry glnd lthough it weighed out hlf of its norml weight. However, n immunohistochemicl exmintion showed tht the numer of FSH- positive cells ws decresed. Therefore, it ws considered tht FSH secretion ws downregulted. A corpus luteum, ut no secondry follicles or mture follicles, ws detected in the ovries, lthough the weights of the ovries were within the norml rnge. The suject seemed to hve een suffering from menorrhe. Amenorrhe, which is one of the criteri used to dignose AN in the DSM-IV (8), iscommoninptients with AN (1). This finding ws in concordnce with her ntemortem sitution ; i.e., she hd een dignosed with AN ut hd stopped receiving tretment. Due to normlities of the hypothlmic-pituitry-thyroid xis, ptients with AN present with condition clled low T3 syndrome, which is chrcterized y low T3 levels, norml or low T4 levels, nd norml TSH levels (9). In the present cse, norml TSH immunorectivity ws detected in the pituitry glnd. In the thyroid, vritions in follicle size, decresed T3 immunorectivity were oserved, ut T4 immunorectivity ws stronger thn tht of T3. These findings suggest tht the suject ws suffering from thyroid dysfunction. As for the function of the hypothlmic-pituitry-drenl xis in AN, AN ptients often exhiit elevted peripherl cortisol levels. Hypercortisolism in AN is considered to e cused y oth incresed cortisol secretion nd decresed cortisol metolism, nd AN ptients disply norml cortisol production (6). In the present cse, the weight of ech drenl glnd hd decresed to the ottom of the norml rnge. In ddition, histologicl exmintion did not detect ny ovious normlities, nd norml cortisol immunorectivity

4 308 A. Ishigmi, et l. Postmortem Dignosis of Anorexi Nervos this cse control HE stining nti-t3 c d nti-t4 e f, c, e ; this cse., d, f ; 40 y.o., femle. The cuse of deth ws pulmonry thromoemolism. Mrked vritions in follicle size were oserved. T3 immunorectivity ws decresed nd T4 immunorectivity ws slightly decresed. Fig. 4 Immunohistochemicl findings of the thyroid glnd, : HE stining, c, d : nti-t3 immunostining, e, f : nti-t4 immunostining Tle 2 Vrious symptoms nd findings tht cn develop s complictions of AN 1. Mentl symptoms depressed mood, socil withdrwl, irritility, insomni, diminished interest in sex 2. Anorml ehvior dietry intke restriction : skipping mels, cutting portion sizes purging : over-exercising ; self-induced vomiting ; misuse of lxtives, diuretics, or enems self-mutiltion, suicidl intent, use of drugs or lcohol 3. Physicl findings weight loss nd mlnutrition hypothermi, edem leukopeni, nemi, thromocytopeni metolic lklosis, hypoklemi, hypochloremi low lood pressure, pulse, or ECG voltge ; sinus rdycrdi ; prolonged QTc intervl hormonl chnges : low serum levels of triiodothyronine (T3) or estrogen low-norml serum levels of thyroxine (T4) menorrhe osteoporosis, frctures, muscle wsting, diminished deep tendon reflex ws oserved in the drenl glnd. Cortisol levels could not e mesured in this cse, ut the ove results suggest tht her cortisol production ws norml. These pthologicl findings indicte tht she hd een suffering with AN until her deth. A recent study reveled tht the fronto-temporo-prietl cortices of AN ptients hd trophied (10). In the present cse, no ovious rin trophiction ws oserved. However, the het generted y the fire might hve ffected the structure of the cererum. AN hs one of the highest mortlity rtes mong psychitric disorders (11). Ftl cses of AN cn involve vrious cuses of deth, including suicide ; lcoholism ; gstric rupturing ; nd the complictions of AN (Tle 2) such s innition, dehydrtion, nd

5 The Journl of Medicl Investigtion Vol. 63 August hert filure (12-14). In our cse, it ws considered tht not the suicide ut the ccidentl fire rought her deth with criminl investigtion y police. Autopsy reveled tht her hed, fce, left lims nd the left side of her trunk were urnt. And the sturtion level of CO-hemogloin in her lood ws 82%, nd soot hd dhered to her irwy nd the upper gstrointestinl trct. No toxicnts except CO were detected, nor ws lcohol. Therefore, it ws considered tht her cuse of deth ws urning with CO intoxiction. 4. CONCLUSION Bsed on the pthologicl findings of the present suject s endocrine orgns, immunohistochemicl exmintions with ntiodies ginst relted hormones might provide informtion tht is useful for dignosing AN, even if the endocrinologicl exmintion from lood cnnot e crried out. 5.CONFLICTS OF INTEREST The uthors hve no conflicts of interest to declre. 6. REFERENCES 1. The Americn Psychitric Assocition : Feeding nd eting disorders. In : Dignostic nd sttisticl mnul of mentl disorders. 5th ed. Wshington DC, 2013, pp Plnning nd Investigtion Committee of the Jpnese Society of Legl Medicine : Questionnire Reserch Performed y the Jpnese Society of Legl Medicine : Weights nd sizes of internl orgns mesured in forensic utopsy cses from 2009 to 2013 in Jpn, http : // 2015, (in Jpnese). 3. Nkmur K, Ymmoto M, Ymzki O, Kwshim Y, Muto K, Somey T, Skuri K, Nozoe S : Prevlence of norexi nervos nd ulimi nervos in geogrphiclly defined re in Jpn. Int J Et Disord 28 : , Gonzlez A, Kohn MR, Clrke SD : Eting disorders in dolescents. Aust Fm Physicin 36 : , Wrren MP : Endocrine mnifesttions of eting disorder. J Clin Endocrinol Met 96 : , Licinio J, Wong ML, Gold PW : The hypothlmic- pituitrydrenl xis in norexi nervos. Psychitry Res 62 : 75-83, Muñoz MT, Argente J : Anorexi nervos in femle dolescents : endocrine nd one minerl density disturnces. Eur J Endocrinol 147 : , The Americn Psychitric Assocition : Eting disorders. In : Dignostic nd sttisticl mnul of mentl disorders. 4th ed. Wshington DC, 1994, pp Kiyohr K, Tmi H, Tkichi Y, Nkgw T, Kumgi LF : Decresed thyroidl triiodothyronine secretion in ptients with norexi nervos : influence of weight recovery. Am J Clin Nutr 50 : , D Agt F, Croppo P, Aminto F, Spltro A, Cglio MM, Bergui M, Lvgnino L, Righi D, Ate- Dg G, Pinessi L, Mortr P, Fssino S : Brin correltes of lexithymi in eting disorders : A voxel-sed morphometry study. Psychitry Clin Neurosci 69 : , Sullivn PF : Mortlity in norexi nervos. Am J Psychitry 152 : , Keel PK, Dorer DJ, Eddy KT, Frnko D, Chrtn DL, Herzog DB : Predictors of mortlity in eting disorders. Arch Gen Psychitry 60 : , Sinicin I, Pnkrtz H, Büttner A, Mll G : Deth due to neurogenic shock following gstric rupture in n norexi nervos ptient. Forensic Sci Int 155 : 7-12, Ppdopoulos FC, Ekom A, Brndt L, Ekselius L : Excess mortlity, cuses of deth nd prognostic fctors in norexi nervos. Br J Psychitry 194 : 10-17, 2009