research article hypovitaminosis d3, leukopenia, and human...

Research ArticleHypovitaminosis D3 Leukopenia and HumanSerotonin Transporter Polymorphism in Anorexia Nervosa andBulimia Nervosa

Anna Tasegian1 Francesco Curcio2 Laura Dalla Ragione3 Francesca Rossetti3

Samuela Cataldi1 Michela Codini1 Francesco Saverio Ambesi-Impiombato2

Tommaso Beccari1 and Elisabetta Albi1

1Department of Pharmaceutical Sciences University of Perugia Via Fabretti No 48 06123 Perugia Italy2Department of Clinical and Biological Sciences University of Udine Piazzale Kolbe No 4 33100 Udine Italy3Department of Eating Disorder Palazzo Francisci Todi USL 1 Umbria Via Cesia No 65 06059 Todi Italy

Correspondence should be addressed to Elisabetta Albi elisabettaalbigmailcom

Received 12 November 2015 Accepted 24 December 2015

Academic Editor Michele Fornaro

Copyright copy 2016 Anna Tasegian et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Vitamin D3 has been described to have different extraskeletal roles by acting as parahormone in obesity diabetes cancercognitive impairment and dementia and to have important regulatory functions in innate immunityThere are no studies showingextraskeletal changes associated with hypovitaminosis D3 in eating disordersMethodsWe have analyzed the blood of 18 patientsaffected by anorexia nervosa and bulimia nervosa collected over a 15-month period We performed a panel of chemical and clinicalanalyses the assay of vitaminD3 the immunoblotting of vitaminD receptor andperoxisomeproliferator-activated receptor gammaand the genotyping of 5-hydroxytryptamine transporter linked polymorphic region ResultsWe choose 18 patients with a normalblood test profile such as thyroid hormones hepatic and renal parameters triglycerides proteins vitamin B12 and folic acidAmong these emerged the case of a woman with long-term anorexia nervosa and the case of a woman with long-term bulimianervosa both complicated by anxiety and depression severe hypovitaminosis D3 decrease of vitamin D receptor leukopenia and5-hydroxytryptamine transporter linked polymorphic region short allele Conclusion The results induce hypothesising that thesevere hypovitaminosis D3might be responsible for the lack of the inflammatory response and the depressive symptoms in patientswith long-term eating disorders

1 Introduction

The eating disorders such as anorexia nervosa (AN) andbulimia nervosa (BN) have been classically described witha clear female preponderance [1] AN is a psychopathologycharacterized by the relentless drive for thinness andor amorbid fear of fatness and BN is characterized by recurrentepisodes of overeating in which large amounts of food areconsumed in short periods [2] Both are often accompaniedby comorbid psychiatric disorders that is affective anxietyand personality disorders [2] Eating disorders have multi-factorial etiology from endocrine abnormalities to geneticpsychological and environment factors [3] Individuals with

AN and BN are consistently characterized by perfectionismobsessive-compulsiveness and dysphoric mood [4]

Vitamin D3 is recognized to have different extraskeletalroles by acting as parahormone Recent evidence correlatesserum vitamin D3 deficiency to obesity diabetes cardiovas-cular risks cancer [5] cognitive impairment and dementia[6] The anti-inflammation activity of vitamin D3 has drawnmore and more attention of researchers to investigate its rolein regulating the progression of inflammatory diseases [7]as an important regulator of innate immunity [8] VitaminD3 deficiency has been associated with inflammatory boweldisease [9] chronic kidney disease [10] asthma and atopy[11] Until now vitamin D3 deficiency in patients with eating

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 8046479 6 pageshttpdxdoiorg10115520168046479

2 Mediators of Inflammation

disorders was correlated only with the risk of osteoporosis[12 13] It is known that vitamin D3 modulates peroxi-some proliferator-activated receptor gamma (PPAR120574) [14]molecule involved in inflammation related to the diet [15 16]

Today is growing acknowledgement that serotonin (5-hydroxytryptamine 5-HT) variations make a substantialcontribution to the pathogenesis of AN and BN Neuroen-docrine rhythms including food intake sleep and reproduc-tive activity together with mood emotion and cognitionare regulated by the midbrain raphe 5-HT system [17] 5-HT is a monoamine neurotransmitter synthesized in thepresynaptic neuron and released during neurotransmissionprocess by presynaptic vesicles into the synaptic cleft thenit binds to receptors on the postsynaptic neuron whereit is metabolised by the monoamine oxidase A enzymeThe 5-HT transporter (5-HTT) resides in the presynapticmembrane allowing reuptake of excess hormone from thesynaptic cleft [18] Relationship between 5-HT dysfunctionand dysregulation of eating behaviour mood and generalpsychopathology was widely reported in AN and BN [419 20] 5-HTT function has impact on early brain devel-opment event-related synaptic plasticity depression andbipolar anxiety obsessive-compulsive schizophrenic andeating disorders and it is a prime target for widely usedantidepressants [17] 5-HTT polymorphism is generated by a44 bp deletion and involves repeat units 6ndash8 and it is locatedclose to a ldquohot spotrdquo for deletion mutagenesis (TGCAGC)in the linked polymorphic region allelic variation in 5-HTT gene plays a role in the expression and modulationof complex traits and behaviour [17] Associations between5-HTT-linked polymorphic region (5-HTTLPR) variationsand eating-disorder subphenotypes were demonstrated [21]In this way 5-HTTLPR deleted (S) allele seems to representa risk factor for eating disorders especially for AN [22]Patrick and Ames reported a regulation of 5-HT synthesisby vitamin D3 in patients with autism [23] attention deficithyperactivity disorder bipolar disorder schizophrenia andimpulsive behaviour [24]

The aim of the work was to highlight the associationamong severe hypovitaminosis D3 reduction of vitamin Dreceptor (VDR) leukopenia and 5-HTTLPR in patients withlong-term AN and BN

2 Methods

21 Ethics Statement All participants provided writteninformed consent prior to inclusion in this project and weretreated in accordance with the Declaration of Helsinki Thestudy protocol and process were assessed and approved bythe Ethics Committee of the Aziende Sanitarie (CEAS) dellaRegione Umbria Italy

22 Patients Blood samples of 18 patients affected by AN(n 11) andor BN (n 7) were collected over a 15-monthperiod (October 2014ndashJanuary 2015) from the Departmentof Eating Disorder Palazzo Francisci (USL 1 Umbria TodiItaly) The population consisted of all females average agewas 40 years (range 14ndash65 yrs) The chemical-clinic analyseswere performed in the diagnostic laboratory of Todi (USL

1 Umbria Italy) Whole blood centrifuged cells and serumwere stored at minus20∘C until used for the assay of vitamin D3immunoblotting and genotyping analysis

23 Materials Standard vitamin D3 was obtained fromSigma Chemical Co (St Louis Missouri USA) anti-peroxisome proliferator-activated receptor gamma (PPAR120574)and anti-vitaminD receptor (VDR) antibodies were obtainedfrom Santa Cruz Biotechnology Inc (California USA)

24 Clinical Measures Participantsrsquo weight and height wereevaluated The levels of thyroid hormones hemochromehepatic and renal parameters iron ferritin cholesteroltriglycerides proteins vitamin B12 and folic acid weremeasured

25 Serum Assays for Vitamin D3 Serum vitamin D3 levelswere measured by LCMSMS method Before the analysisthe sample was prepared by adding 150 120583L of precipitationreagent containing Internal Standard to 50 120583L of patientserum or quality control serum after 10 minutes of incuba-tion 50120583L of supernatant was injected The signal from theanalytes wasmeasured against the calibration curve using theMultiQuant 21 software For the analysis Shimadzu UFLC-XR system Shimadzu Kyoto Japan equipped with a DGU-20A3 degasser LC-20AD XR binary pump SIL-20ACXRautosampler CTO-20AC column oven and a communica-tion bus module CBM-20A was used The UFLC systemwas connected to a 4000 QTRAP linear ion trap mass spec-trometer (AB Sciex Concord ON Canada) equipped withan APCI source operated in the positive ionization modeA 6-port automatic switching valve was interposed betweenthe chromatographic system and the mass spectrometer Thecontroller software was Analyst version 16 The limit ofdetection (LLOD) for this assay was 067120583gL The low limitof quantification (LLOQ) was 225 120583gL and the linearity was225ndash250120583gL CVs were 6 at various concentrations acrossthe analytical measurement range

26 Electrophoresis and Western Blot Analysis The analysiswas performed as previously reported [25] 30 120583g of proteinwas used for SDS-PAGE electrophoresis in 10 polyacry-lamide slab gel Proteins were transferred into nitrocellulosefor 90min and the membranes were blocked for 30minwith 05 no fat-dry milk in PBS pH 75 and incubatedovernight at 4∘C with antibody anti-PPAR120574 or VDR Theblots were incubated with horseradish-conjugated secondaryantibodies for 90min The enhanced chemiluminescence(ECL) reaction was performed with the ECL kit from Amer-sham (Rainham Essex UK) Immunoblots of proteins werereprobed after stripping The apparent molecular weight ofthe proteins was calculated according to the migration ofmolecular size standard The area density of the bands wasevaluated by densitometry scanning and analyzed with ScionImage

27 Genotyping of 5-HTTLPR Genomic DNA (gDNA)was extracted from whole blood samples using QIAampregDNA Mini kit (QIAGEN) Genotyping of the 5-HTTLPR

Mediators of Inflammation 3

Table 1 Parameters considered for the study ED = eating disorder TO = time of onset (years) VD3 = vitamin D3 (16ndash60 ngmL normalvalues) WBC = white blood cells (36ndash96 normal values) N = neutrophils (42ndash75 19 times 103ndash80 times 103 normal values) L = lymphocytes(205ndash511 09 times 103ndash35 times 103 normal values) I = iron (55ndash150 120583gdL normal values) F = ferritin (11ndash307120583gdL normal values) T =transferring (180ndash360 120583gdL normal values) C = cholesterol (130ndash220mgdL normal values) G = glycemia (60ndash110mgdL normal values)

N∘ Age ED TO VD3 WBC N L I F T C G1 40 BN lt10 104 86 79 150 64 50 207 166 722 18 AN lt10 270 438 378 520 51 748 203 139 773 19 BN lt10 350 775 705 209 126 305 313 144 854 41 AN lt10 186 1429 677 254 166 63 294 190 845 16 BN lt10 133 419 537 365 140 599 254 146 766 55 BN lt10 140 432 56 343 91 457 231 165 907 16 AN lt10 220 427 604 288 59 607 212 110 708 40 AN lt10 254 599 491 425 119 590 272 170 729 23 BN lt10 181 469 661 225 85 188 242 163 7210 18 AN lt10 195 466 469 432 94 1835 211 147 10311 15 AN lt10 175 470 600 312 98 54 208 213 7712 18 BN lt10 271 678 679 227 40 416 245 158 7113 29 AN 15 360 291 544 366 67 431 248 158 8214 14 AN lt10 207 437 570 32 128 628 215 160 7615 65 AN lt10 300 662 660 226 70 813 266 157 7416 44 BN 26 400 321 640 170 82 475 212 171 8917 18 AN lt10 316 481 479 407 48 658 262 226 8018 18 AN lt10 275 447 411 480 60 216 219 133 72

polymorphism was performed by polymerase chain reactionusing the PhusionHot Start II High FidelityDNAPolymerase(Thermo Scientific) The following primer pair was used51015840-GGCGTTGCCGCTCTGAATGC-31015840 (primer forward) 51015840-GAGGGACTGAGCTGGACAACCAC-31015840 (primer reverse)[26 27] The amplification of sim50 ng of gDNA was carriedout after 3010158401015840 of initial denaturation at 98∘C for 35 cycles(denaturation at 98∘C for 1510158401015840 annealing at 69∘C for 3010158401015840 andextention at 72∘C for 1510158401015840) followed by a final extention at72∘C for 51015840 10 120583L aliquots of each PCR product were resolvedon 25 agarose gel electrophoresis in 1x TBE buffer at 90Volts Genotype was determined by fragment sizes 484 bp (Sshort allele) and 528 bp (L long allele) 100 bp SHARPMASS(Euroclone) was used as DNA ladder

28 Statistical Analysis Three experiments performed induplicate were performed for each analysis Data areexpressed as mean plusmn SD and 119905-test was used for statisticalanalysis

3 Results and Discussion

31 Results AN (n 11) and BN (n 7) patients were selected tohave normal blood test profile as thyroid hormones hepaticand renal parameters triglycerides proteins vitamin B12and folic acid Thus 18 patients were enrolled

To highlight a possible association with immunity cellsand to exclude defects of intestinal absorption andor ofmetabolic disorders the concentration of vitamin D3 wascorrelated to the total white blood cells (WBC) neutrophils(N) lymphocytes (L) iron ferritin transferrin cholesterol

and glycemia (Table 1) All patients had normal levels offerritin transferrin and glycemia and only 3 patients showedlow levels of iron and 1 patient low level of cholesterol withoutchanges of vitamin D

Of 18 patients 13 presented normal levels of vitamin D3between 16 and 60 ngmL [28] (group A) and 5 patients werevitamin D3-deficient We chose to divide patients with lowlevels of vitamin D3 in 2 groups patients with 10ndash16 ngmLvitamin D3 (group B patients 1 5 and 6) and patients with1ndash4 ngmL (group C patients 13 16)

The patients of groups A and B showed no changes inWBC only patient n 2 presented reduction of N and increaseof L as a sign of possible chronic inflammation (Table 1)Interestingly patients of group C had low level of total WBCpatient 13 low level of N (159 times 103) and patient 16 low levelof L (071 times 103) These data could suggest therefore theexistence of a relation between the low level of vitamin D3and the WBC disorders

It is important to note that there was no correlationbetween the concentration of vitamin D3 and age In factof 18 patients 10 were found to have an age in the range of10ndash20 years 2 in the range of 21ndash30 years 2 in the range of31ndash40 years and 2 in the range of 41ndash50 years and 1 patientwas found to have an age in the range of 51ndash61 years and 1in the range of 61ndash70 years (Figure 1(a)) Patients with lowconcentration of vitamin D3 had 16 40 and 55 years andpatients with very low level of vitaminD3 had 29 and 44 years(Figure 1(a))

Differently a relation between level of vitamin D3 andtime of onset of the eating disorders was evident In factof 18 patients 16 (89) had a time of onset under 10 years


Age

1 vit D L

1 vit D VL

1 vit D L

1 vit D L

1 vit D VL

31ndash4021ndash3010ndash20


(a)

89

11

Time of onset

19 vit D L

gt10

lt10

100 vit D VL

(b)

Figure 1 Level of vitamin D3 in patients affected with anorexia nervosa and bulimia nervosa The data were analysed in relation to (a) theage and (b) time of the onset Vit D L low level vitamin D3 Vit D VL very low level of vitamin D3

and included patients with normal level and low level ofvitamin D3 whereas 2 patients (11) had a very long historyof disease one with AN (patient n 13) and one with BN(patient n 16) and both had very low level of vitamin D3(Table 1 and Figure 1) The results suggested the importanceof blood concentration of vitamin D3 in patients who had avery long history on eating disorders but remained unknownif these patients had also deficiency of VDR in the blood cells

To address this question the immunoblotting analysiswas performed with a specific antibody against vitamin D3receptor (VDR) in patients n 13 (experimental 1 Ex1) and n16 (experimental 2 Ex2) Patient number 11 with normal levelof vitamin D3 (control 1 C1) and patient number 5 with lowlevel of vitamin D3 (control 2 C2) both with similar bloodtest profile were chosen as controls

The results highlighted that the band density of VDRcorresponding to 48 kDa apparent molecular weight was lesscolored in Ex samples than in C samples (Figure 2(a)) Theanalysis of band density showed that VDR of Ex patientswas significantly lower than that present in C patients(Figure 2(b)) In particular the VDR Ex1 was reduced 33and Ex2 55 with respect to that of C patients whose valuewas very similar

Since PPAR is regulated by vitamin D we choose totest PPAR expression by immunoblotting to evaluate anactivation of the inflammatory pathway related to the diet Nochanges in protein level were found (Figures 2(a) and 2(b))

The two experimental patients with very low level ofvitamin D3 and reduction of VDR had family problemsnegativistic and depressive personality disturbances It wasdemonstrated that 5-HTTLPR had an impact on the develop-ment of eating-disorder symptoms [29] and thus we studied5-HTTLPR in C and Ex patients chosen for the study Ourresults demonstrated the presence of 5-HTTLPR variantshort allele (S) in both Ex samples with a very long history

of AN (Ex1) and BN (Ex2) that was absent in C patients(Figure 3)

32 Discussion We herein reported the case of a womanwith long-term AN and the case of a woman with long-termBN complicated by very low level of vitamin D decrease ofVDR leukopenia and the S allele of the 5-HT transporterpolymorphism This information emerged from a study of18 AN (n 11) and BN (n 7) patients without blood testprofile that suggested endocrine metabolic or absorptiondisorders Because the goal of the work was to highlightassociations of very low amount of vitamin D3 with otherparameters in patients with eating disorders as controls wechose two patients enrolled in the same study one with ANwho had normal levels of vitamin D3 and one with BNwho had slightly low value of vitamin D It was known thatvitamin D3 deficiency increases the risk for acquiring severalinfectious diseases [30] since it had effects on innate andadaptive immunity and has antimicrobial anti-inflammatoryand immunomodulatory functions [31] Our data showedthat the only two patients who had severe hypovitaminosispresented reduction of WBC At the moment we do notknow if these patients had a defect of immunity response Itwill be interesting in the future to investigate the associationof immunodeficiencies and severe hypovitaminosis D3 inAN and BN patients It could be very important becausewe showed that the blood cells of the two patients withsevere hypovitaminosis D3 had a reduced content of the VDRand T cells have been shown to express the VDR and tobe both direct and indirect targets of vitamin D3 [32] Wehave recently demonstrated that VDR is located in nuclearlipid microdomains that act as platform for active chromatinanchoring so that vitamin D3 can regulate gene expression[33] So we speculate on the possibility that the very low levelof vitamin D3 and the reduction of VDR in blood cells might


VDR

C1 Ex2Ex1C2

55kDa

48kDa

PPAR120574

(a)

VDR

C2 Ex1 Ex2C10

10000

20000

30000

40000

Are

a den

sity

PPAR120574

lowast

lowast

(b)

Figure 2 Immunoblotting of vitamin D receptor (VDR) andperoxisome proliferator-activated receptor gamma (PPAR120574) (a)Immunoblot of proteins was probed with specific antibodies andvisualized by ECL in a patient with anorexia nervosa and normallevel of vitamin D3 (C1) a patient with bulimia nervosa and slightlyreduced level of vitamin D3 (C2) a patient with anorexia nervosaand very low level of vitamin D3 (Ex1) and a patient with bulimianervosa and very low level of vitamin D3 (Ex2) Apparent molecularweight was calculated according to the migration of molecular sizestandards (b) The area density was calculated with Scion Imageprogramme on densitometry scanning the data represent the meanplusmn SD of three experiments performed in duplicate (Significancelowast

119875 lt 0001 versus C1 and C2 sample)

be responsible for the S allele of the 5-HTT polymorphismthat was described to be related to eating disorder as reportedabove [21] On the other hand the 5-HTTLPR S allele hasbeen associatedwith differential susceptibility for anxiety anddepression in multiple psychiatric disorders [34] and the twoAN and BN patients under study presented these symptomsin their personal history It could be due to themodification ofthe serotonergic systems that support emotion by 5-HTTLPRS allele that induced reduction of gene and protein expression[17] In this way the transporter function was less efficientand consequently serotonin remained in the synapse longerand was recycled more slowly resulting in a net reduction incirculating serotonin [17]

4 Conclusions

The most prominent feature of the study was the identifi-cation of a woman with long-term AN and a woman withlong-term BN with severe hypovitaminosis D3 This studyprovides a unique insight into the association among the verylow level of vitamin D3 the decrease of VDR leukopenia

Ladder C1 Ex2Ex1C2

L alleleS allele

1000

300

500

400

600700800900

100 bp

Figure 3 Agarose gel electrophoresis of 5-HTTLPR polymorphismamplified by PCR The two patients with very low level of vitaminD3 E1 and E2 bear the short allele (S) The two control patients areinstead homozygous for the long allele (L) The molecular weight ofthe corresponding bands is calculated according to the migration ofmolecular size standards (see Section 2)

and 5-HTTLPR extended to emotional dysfunction Theresults induce hypothesising that the severe hypovitaminosisD3 might be responsible for lack of inflammatory responseand reduction in mood in patients with long-term eatingdisorders The results could be useful to use vitamin D3as anti-inflammatory molecule for novel therapies in thetreatment of eating disorders

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The study was supported by University of Udine ItalyAssociazione ldquoMi Fido di Terdquo ONLUS and Fondazione Cassadi Risparmio di Citta di Castello Italy

References

[1] A EAndersen andG L Ryan ldquoEating disorders in the obstetricand gynecologic patient populationrdquoObstetrics andGynecologyvol 114 no 6 pp 1353ndash1367 2009

[2] P Sharan and A S Sundar ldquoEating disorders in womenrdquo IndianJournal of Psychiatry vol 57 no 6 supplement 2 pp S286ndashS2952015

[3] K M Culbert S E Racine and K L Klump ldquoWhat we havelearned about the causes of eating disordersmdasha synthesis ofsociocultural psychological and biological researchrdquo Journal ofChild Psychology and Psychiatry vol 56 no 11 pp 1141ndash11642015


[4] W Kaye ldquoNeurobiology of anorexia and bulimia nervosardquoPhysiology amp Behavior vol 94 no 1 pp 121ndash135 2008

[5] B Matyjaszek-Matuszek M Lenart-Lipinska and EWozniakowska ldquoClinical implications of vitamin D deficiencyrdquoPrzegląd Menopauzalny vol 14 no 2 pp 75ndash81 2015

[6] A Banerjee V K Khemka A Ganguly D Roy U Ganguly andS Chakrabarti ldquoVitamin D and Alzheimerrsquos disease neurocog-nition to therapeuticsrdquo International Journal of AlzheimerrsquosDisease vol 2015 Article ID 192747 11 pages 2015

[7] Z Lin and W Li ldquoThe roles of vitamin D and its analogs ininflammatory diseasesrdquo Current Topics in Medicinal Chemistryvol 15 pp 1ndash20 2015

[8] V Dimitrov and J H White ldquoSpecies-specific regulation ofinnate immunity by vitamin D signalingrdquoThe Journal of SteroidBiochemistry and Molecular Biology 2015

[9] R Del Pinto D Pietropaoli A K Chandar C Ferri and FCominelli ldquoAssociation between inflammatory bowel diseaseand vitaminD deficiencyrdquo Inflammatory Bowel Diseases vol 21no 11 pp 2708ndash2717 2015

[10] W-C Liu C-M Zheng C-L Lu et al ldquoVitaminDand immunefunction in chronic kidney diseaserdquo Clinica Chimica Acta vol450 pp 135ndash144 2015

[11] E Morales M Sanchez-Solis and L Garcia-Marcos ldquoVitaminD metabolism genes in asthma and atopyrdquo Mini-Reviews inMedicinal Chemistry vol 15 no 11 pp 913ndash926 2015

[12] D Modan-Moses Y Levy-Shraga O Pinhas-Hamiel et alldquoHigh prevalence of vitamin D deficiency and insufficiency inadolescent inpatients diagnosed with eating disordersrdquo Interna-tional Journal of Eating Disorders vol 48 no 6 pp 607ndash6142015

[13] M Misra and A Klibanski ldquoBone health in anorexia nervosardquoCurrent Opinion in Endocrinology Diabetes and Obesity vol 18no 6 pp 376ndash382 2011

[14] T-I Lee Y-H Kao Y-C Chen W-C Tsai C-C Chungand Y-J Chen ldquoCardiac metabolism inflammation and per-oxisome proliferator-activated receptors modulated by 125-dihydroxyvitamin D

3

in diabetic ratsrdquo International Journal ofCardiology vol 176 no 1 pp 151ndash157 2014

[15] W X Liu T Wang F Zhou et al ldquoVoluntary exercise preventscolonic inflammation in high-fat diet-induced obese mice byup-regulating PPAR-120574 activityrdquo Biochemical and BiophysicalResearch Communications vol 459 no 3 pp 475ndash480 2015

[16] J H Ooi Y Li C J Rogers and M T Cantorna ldquoVitamin Dregulates the gut microbiome and protects mice from dextransodium sulfate-induced colitirdquo Journal of Nutrition vol 143 no10 pp 1679ndash1686 2013

[17] A Heils A Teufel S Petri et al ldquoAllelic variation of humanserotonin transporter gene expressionrdquo Journal of Neurochem-istry vol 66 no 6 pp 2621ndash2624 1996

[18] M Sadkowski B Dennis R C Clayden et al ldquoThe role ofthe serotonergic system in suicidal behaviourrdquoNeuropsychiatricDisease and Treatment vol 9 pp 1699ndash1716 2013

[19] W H Kaye H E Gwirtsman D T George and M H EbertldquoAltered serotonin activity in anorexia nervosa after long-term weight restoration does elevated cerebrospinal fluid 5-hydroxyindoleacetic acid level correlate with rigid and obses-sive behaviorrdquo Archives of General Psychiatry vol 48 no 6 pp556ndash562 1991

[20] P Monteleone F Brambilla F Bortolotti and M Maj ldquoSero-tonergic dysfunction across the eating disorders relationshipto eating behaviour purging behaviour nutritional status and

general psychopathologyrdquo Psychological Medicine vol 30 no 5pp 1099ndash1110 2000

[21] H Steiger ldquoEating disorders and the serotonin connectionstate trait and developmental effectsrdquo Journal of Psychiatry andNeuroscience vol 29 no 1 pp 20ndash29 2004

[22] R Calati D De Ronchi M Bellini and A Serretti ldquoThe 5-HTTLPR polymorphism and eating disorders ameta-analysisrdquoInternational Journal of Eating Disorders vol 44 no 3 pp 191ndash199 2011

[23] R P Patrick and B N Ames ldquoVitamin D hormone regulatesserotonin synthesis Part 1 relevance for autismrdquo The FASEBJournal vol 28 no 6 pp 2398ndash2413 2014

[24] R P Patrick and B N Ames ldquoVitamin D and the omega-3 fatty acids control serotonin synthesis and action Part 2Relevance for ADHD bipolar disorder schizophrenia andimpulsive behaviourrdquo The FASEB Journal vol 29 no 6 pp2207ndash2222 2015

[25] E Bartoccini FMarini E Damaskopoulou et al ldquoNuclear lipidmicrodomains regulate nuclear vitamin D

3

uptake and influ-ence embryonic hippocampal cell differentiationrdquo MolecularBiology of the Cell vol 22 no 17 pp 3022ndash3031 2011

[26] A Payton L Gibbons Y Davidson et al ldquoInfluence of sero-tonin transporter gene polymorphisms on cognitive declineand cognitive abilities in a non demented elderly populationrdquoMolecular Psychiatry vol 10 no 12 pp 1133ndash1139 2005

[27] J Chen Q Kang W Jiang et al ldquoThe 5-HTTLPR conferssusceptibility to anorexia nervosa in Han Chinese evidencefrom a case-control and family-based studyrdquo PLoS ONE vol 10no 3 Article ID e0119378 2015

[28] M Holick ldquoVitamin Drdquo in Modern Nutrition in Healthand Disease A C Ross Ed pp 1ndash1616 Wolters KluwerHealthLippincott Williams ampWilkins 2014

[29] K Akkermann K Kaasik E Kiive N Nordquist L Orelandand J Harro ldquoThe impact of adverse life events and theserotonin transporter gene promoter polymorphism on thedevelopment of eating disorder symptomsrdquo Journal of Psychi-atric Research vol 46 no 1 pp 38ndash43 2012

[30] R RWatkins T L Lemonovich andRA Salata ldquoAnupdate onthe association of vitaminDdeficiencywith common infectiousdiseasesrdquo Canadian Journal of Physiology and Pharmacologyvol 93 no 5 pp 363ndash368 2015

[31] A S Amaya-Mejıa P M OrsquoFarrill-Romanillos L V Galindo-Pacheco et al ldquoVitamin D deficiency in patients with commonvariable immunodeficiency with autoimmune diseases andbronchiectasisrdquo Revista Alergia Mexico vol 60 no 3 pp 110ndash116 2013

[32] J Chen D Bruce and M T Cantorna ldquoVitamin D receptorexpression controls proliferation of naıve CD8+ T cells anddevelopment of CD8 mediated gastrointestinal inflammationrdquoBMC Immunology vol 15 article 6 2014

[33] G Cascianelli M Villani M Tosti et al ldquoLipid microdomainsin cell nucleusrdquoMolecular Biology of the Cell vol 19 no 12 pp5289ndash5295 2008

[34] J R Homberg and K-P Lesch ldquoLooking on the bright side ofserotonin transporter gene variationrdquo Biological Psychiatry vol69 no 6 pp 513ndash519 2011

Submit your manuscripts athttpwwwhindawicom

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


disorders was correlated only with the risk of osteoporosis[12 13] It is known that vitamin D3 modulates peroxi-some proliferator-activated receptor gamma (PPAR120574) [14]molecule involved in inflammation related to the diet [15 16]

Today is growing acknowledgement that serotonin (5-hydroxytryptamine 5-HT) variations make a substantialcontribution to the pathogenesis of AN and BN Neuroen-docrine rhythms including food intake sleep and reproduc-tive activity together with mood emotion and cognitionare regulated by the midbrain raphe 5-HT system [17] 5-HT is a monoamine neurotransmitter synthesized in thepresynaptic neuron and released during neurotransmissionprocess by presynaptic vesicles into the synaptic cleft thenit binds to receptors on the postsynaptic neuron whereit is metabolised by the monoamine oxidase A enzymeThe 5-HT transporter (5-HTT) resides in the presynapticmembrane allowing reuptake of excess hormone from thesynaptic cleft [18] Relationship between 5-HT dysfunctionand dysregulation of eating behaviour mood and generalpsychopathology was widely reported in AN and BN [419 20] 5-HTT function has impact on early brain devel-opment event-related synaptic plasticity depression andbipolar anxiety obsessive-compulsive schizophrenic andeating disorders and it is a prime target for widely usedantidepressants [17] 5-HTT polymorphism is generated by a44 bp deletion and involves repeat units 6ndash8 and it is locatedclose to a ldquohot spotrdquo for deletion mutagenesis (TGCAGC)in the linked polymorphic region allelic variation in 5-HTT gene plays a role in the expression and modulationof complex traits and behaviour [17] Associations between5-HTT-linked polymorphic region (5-HTTLPR) variationsand eating-disorder subphenotypes were demonstrated [21]In this way 5-HTTLPR deleted (S) allele seems to representa risk factor for eating disorders especially for AN [22]Patrick and Ames reported a regulation of 5-HT synthesisby vitamin D3 in patients with autism [23] attention deficithyperactivity disorder bipolar disorder schizophrenia andimpulsive behaviour [24]

The aim of the work was to highlight the associationamong severe hypovitaminosis D3 reduction of vitamin Dreceptor (VDR) leukopenia and 5-HTTLPR in patients withlong-term AN and BN

2 Methods

21 Ethics Statement All participants provided writteninformed consent prior to inclusion in this project and weretreated in accordance with the Declaration of Helsinki Thestudy protocol and process were assessed and approved bythe Ethics Committee of the Aziende Sanitarie (CEAS) dellaRegione Umbria Italy

22 Patients Blood samples of 18 patients affected by AN(n 11) andor BN (n 7) were collected over a 15-monthperiod (October 2014ndashJanuary 2015) from the Departmentof Eating Disorder Palazzo Francisci (USL 1 Umbria TodiItaly) The population consisted of all females average agewas 40 years (range 14ndash65 yrs) The chemical-clinic analyseswere performed in the diagnostic laboratory of Todi (USL

1 Umbria Italy) Whole blood centrifuged cells and serumwere stored at minus20∘C until used for the assay of vitamin D3immunoblotting and genotyping analysis

23 Materials Standard vitamin D3 was obtained fromSigma Chemical Co (St Louis Missouri USA) anti-peroxisome proliferator-activated receptor gamma (PPAR120574)and anti-vitaminD receptor (VDR) antibodies were obtainedfrom Santa Cruz Biotechnology Inc (California USA)

24 Clinical Measures Participantsrsquo weight and height wereevaluated The levels of thyroid hormones hemochromehepatic and renal parameters iron ferritin cholesteroltriglycerides proteins vitamin B12 and folic acid weremeasured

25 Serum Assays for Vitamin D3 Serum vitamin D3 levelswere measured by LCMSMS method Before the analysisthe sample was prepared by adding 150 120583L of precipitationreagent containing Internal Standard to 50 120583L of patientserum or quality control serum after 10 minutes of incuba-tion 50120583L of supernatant was injected The signal from theanalytes wasmeasured against the calibration curve using theMultiQuant 21 software For the analysis Shimadzu UFLC-XR system Shimadzu Kyoto Japan equipped with a DGU-20A3 degasser LC-20AD XR binary pump SIL-20ACXRautosampler CTO-20AC column oven and a communica-tion bus module CBM-20A was used The UFLC systemwas connected to a 4000 QTRAP linear ion trap mass spec-trometer (AB Sciex Concord ON Canada) equipped withan APCI source operated in the positive ionization modeA 6-port automatic switching valve was interposed betweenthe chromatographic system and the mass spectrometer Thecontroller software was Analyst version 16 The limit ofdetection (LLOD) for this assay was 067120583gL The low limitof quantification (LLOQ) was 225 120583gL and the linearity was225ndash250120583gL CVs were 6 at various concentrations acrossthe analytical measurement range

26 Electrophoresis and Western Blot Analysis The analysiswas performed as previously reported [25] 30 120583g of proteinwas used for SDS-PAGE electrophoresis in 10 polyacry-lamide slab gel Proteins were transferred into nitrocellulosefor 90min and the membranes were blocked for 30minwith 05 no fat-dry milk in PBS pH 75 and incubatedovernight at 4∘C with antibody anti-PPAR120574 or VDR Theblots were incubated with horseradish-conjugated secondaryantibodies for 90min The enhanced chemiluminescence(ECL) reaction was performed with the ECL kit from Amer-sham (Rainham Essex UK) Immunoblots of proteins werereprobed after stripping The apparent molecular weight ofthe proteins was calculated according to the migration ofmolecular size standard The area density of the bands wasevaluated by densitometry scanning and analyzed with ScionImage

27 Genotyping of 5-HTTLPR Genomic DNA (gDNA)was extracted from whole blood samples using QIAampregDNA Mini kit (QIAGEN) Genotyping of the 5-HTTLPR















Age

1 vit D L

1 vit D VL

1 vit D L

1 vit D L

1 vit D VL



(a)

89

11

Time of onset

19 vit D L

gt10

lt10

100 vit D VL

(b)










VDR

C1 Ex2Ex1C2

55kDa

48kDa

PPAR120574

(a)

VDR

C2 Ex1 Ex2C10

10000

20000

30000

40000

Are

a den

sity

PPAR120574

lowast

lowast

(b)




4 Conclusions


Ladder C1 Ex2Ex1C2

L alleleS allele

1000

300

500

400

600700800900

100 bp





Acknowledgments


References
















3














3
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Age

1 vit D L

1 vit D VL

1 vit D L

1 vit D L

1 vit D VL



(a)

89

11

Time of onset

19 vit D L

gt10

lt10

100 vit D VL

(b)










VDR

C1 Ex2Ex1C2

55kDa

48kDa

PPAR120574

(a)

VDR

C2 Ex1 Ex2C10

10000

20000

30000

40000

Are

a den

sity

PPAR120574

lowast

lowast

(b)




4 Conclusions


Ladder C1 Ex2Ex1C2

L alleleS allele

1000

300

500

400

600700800900

100 bp





Acknowledgments


References
















3














3
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VDR

C1 Ex2Ex1C2

55kDa

48kDa

PPAR120574

(a)

VDR

C2 Ex1 Ex2C10

10000

20000

30000

40000

Are

a den

sity

PPAR120574

lowast

lowast

(b)




4 Conclusions


Ladder C1 Ex2Ex1C2

L alleleS allele

1000

300

500

400

600700800900

100 bp





Acknowledgments


References
















3














3
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























3














3
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article hypovitaminosis d3, leukopenia, and human...

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