abstract - aup.edu.phaup.edu.ph/alumni/wp-content/uploads/r17.pdftype of alternative therapy called...
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Ancient cultures.according to theWorld Research
Foundation. believed fasting-a~iaJ or total abstention ffomall foods. or a select abstentionfrom prohibited foods - couldpurify the soul. A growingnumber of practitioners of a
type of alternative therapycalled metabolic therapy asstudied by Chill, Bianchi.Franchi-Gazzola & Bussolatiin 2012 believe thaI the bodvhas environmental toxins anaother harmful substances thatcan be removed by fastingor detoxifying the body.
I. INTRODUCTION
The ability of the immune system to effectively protect the bodyfrom infection depe-nds on various factors, one of which is properdiet. Fasting. the practice of withholding food for a period oftime
bas been practiced allover the world mainly as part of a religious ritual. Themain emphasis of this study is to elucidate the effects of fasting on the humaninnate immunity, This re-search aims to investigate whether fasting would causea significam chililge ill the neutrophilic phagocytic activity during a 24-hourreligious fast. The study was performed on 20 healthy students who fasted for14-hours. Blood samples were collected before and after the fasting period andanalyzed for white blood cell count and phagocytic activity, The paired J-Testcomparing before and after fasting values. showed lhal (here is a signiticomincrease in all of the measured parameters for phagocytosis constituting anaverage of 54.14% increase on the overall activity or neutrophits after thefasting period. Statistical analyses provided no evidence thai the change inwhite blood cell count is related to the phagocytic activity which leads to theconclusion thai an increase in phagocytic index is associated with enhancedfunction rather than the decrease of number of leukocytes.
Abstract
Alain Justin Berbano, Richardson Delas Alas,David Hendrik Putra Palali Ma. Estrella H. Sales, RMT
EFFECTS OF 24-HOUR FASTING ON THE [N VITROPHAGOCYTIC ACT[vITY OF NEUTROPH£LS
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A study conducted by Chia Wei etaL (2014) on the effects of prolonged fasting(48 - 110 hours of fasting) on the immunesystem of mice that were administered withchemotherapeutic drugs. The study showedthat cycles of prolonged fasting protectedhematopoietic cells from chemotoxidty andinduced immune system regeneration, shifting stem cells&om a dormant state to a staleof self-renewal to reverse immunosuppression, Prolonged iasting also lowered levels ofIGF-I. a growth-factor hormone that Longoand others have linked to aging. tumor progression and cancer riskMeasurement of neutrophil parameters is anarea of interest in immunology because neutrophils playa critical role in host defense,Neutrophils constitute an organisms firstline of defense against external aggressionand represent one of the key nonspecifichost defense cell populations responsible forthe phagocytosis of many microbial. bacterial, fungal and viral pathogens, (Stevens,2010) Phagocytosis is defined as the ingestion of particles by cells, and this process involv... the binding of (opsonized) particles10 the surface of phagocytic cells. followedby the internalization and destruction ofthese particles, The killing and microbicidal functions of neutrophils are facilitatedby the metabolic pathways involving theactivation of NADPH oxidase system andthe myeloperoxidase (MPO)_ (Todar, 2oo8)Neutrophils are also known to be involvedin the synthesis and release of immunomodulatory cytokines that influence both T celland Bcell activities- (Pyne, t994)
However, fasting is not only practiced for health reasons. Almost all majorreligions of the world have a form of fastingincorporated in their beliefs (Brown & Mussell, 1984). While religious fast is partakenprimarily for spiritual purposes. it also hasthe potential to greatly affect one's physicalhealth (Desai, 2oo0). Accordingly. the healtheffects of religious fasting have recently beenthe subject of scientific inquiry. with mostof the research being performed measuringhealth parameters during Ramadan. a timeperiod in whkh Muslim pilgrims subjectthemselves to a partial fast wherein me-alsare only taken at the start and the end of aday thereby inducing a fast lasting for an average of 12hours/day for 40 days (latifynia,Vojgani,Ghargozlon & Sharifian, 2oo9)A cross sectional study by Khazaei, Bokaeian & jalili in 2013 involving 90 athletesduring the month of Ramadan showed apositive increase ofC4 and IgAlevelsamongthe participants. TIle increase in C4 and IgAdemonstrated protective effects on an individual's immune system against infection. Inanother study by Hiramoto et, al, in 2008.it has also been observed that a nutritionalstress of a )6+hour fast increased the number of neutrophils in the peripheral blond inboth the elderly and young adult subjects,
They claim that fasting allows the body tofocus energy on cleansing and healing itself. According (0 these practitioners. fasting helps the immune system work moreefficiently. nlJo\villg more oxygen and whiteblood cells to 60\\1 dlfOUgh the body, he1pthe body burn more fat, help increase energy. and allows other healing functions toimprove. A study by the University of Berlin(20)3) on new therapeutic approach 10 fighlcancer revealed similar results.
Effedl of l.f..Hotlr Fastitlg oa the YiI,-oPh.agocrtir :\(tiYtly or NflltropbUs
Phagocyttc Index [\,aI113[:100Blood samples from the subjects
were collected l-hour postprandial for thenon-fasting state, and a 24-hour fastingblood specimen for the fasting state. Eachsubject served 3S his own control and the2-hour post prandial value was the controlfor each subject Blood samples were collected in heparin rubes for the phagocyticassay. and EDTA lubes for compete bloodcount (CBC).
A sample of 0.9 mJ freshly drawnheparinized blood was mixed in siliconized tubes and stoppers with 0.1 ml of theopsonized bacterial suspension of Staphylococcus aureus. This was rotated mechanicallyend to end at 370C for 30 minutes.Blood smears were prepared on glass slides.(Latifynia et. aJ. 2000 and Heir, B. 2001)Prepared blood smears from the patientswere stained with \Vright stain. Cells werecounted under oil-immersion. and 40 ceJJswere counted to obtain a reliable result.
ning of the study, The group of subjects included in this study consisted of 20 healthyAUP college students (5 females and ISmales). The selected group was ideal in thisstudy for two reasons. (I) All of the subjectsare Christians with the majority being Sevemh-day Adventists and were committed toobserving a religious fast during the testingperiod. This enabled the research to obtainresults from this event. (2) Since all the participants are students enrolled in AUP andresiding in the dormitories, it can be considcreel that the similarities in their demogmphics like diet. levels of stress. physical exertion may have limited possible confoundingfactors.
II. MATERJALSANDMETHODSampling
This study was approved by the Department of Medical Laboratory Science ofthe Adventist Universiry of the Philippines(AUP). and written informed consent wasobtained from all subjects before the begin-
Since most of the study centers onthe.effects of prolonged fasting on the immune system, this study explored the effectsof24-hour fasting on one of the major functions of human innate immunity - neutrophil phagocytosis.
Like every other cellular funclion. phagocytosis is an energy requiring mechanisrnthat is inhibited by an inadequate supply ofglucose (Segal. 2005). However. it has alsobeen noted thai excessive glucose in theblood may also decrease phagocytic activity(Van Oss. 1971). This phenomenon can beobserved in patients with poorly managedcases of diabetes mellitus, OM patients arecharacterized by elevated blood glucoselevels and lowered resistance 10 infection.Diabetes mellitus patients present physiological impairments. including diminishedimmunological function and inftnllunaloryresponses (chemotaxis. phagocytosis andk-illing). leading 10 higher susceptibility 10bacterial and fungal infections. (Hotamisligil, 2(06) Studies done by Wilson & Ree vesin 1986_ Alba-Loureiro et al in 2006 &Kempf et. al in 2007 have suggested that apossible cause of these weaker immune responses is neutrophil dysfunction caused byhyperglycemia, Similar findings have beenobserved in obese animal models. (Nabi, Islam, Rahman & Biswas. 2005: Slavov, Dzhelebov, Andonova & Girginov. 2009 & DeSourza Ferreira. 20 I 2)
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111e study papulation, aged 19-27years old. consisted of l; male and 5 femalecollege students frornAUP. Twenty-six people volunteered for the study but due to various limitations and circumstances (including the lack of transportal ion, volunteerswith CBC parameters outside the normalrange and volunteers that prematurely broketbeir fast) some blood samples were considered unsuitable, leaving only 20 volunteerseligible for study.For statistical analyses, a paired t-test wasused 10 compare the phagocytic index.phagocyiic percent and phagocytic activity
Ill. RESULTS
Complete Blood CountsComplete Blood Count was deter
mined using Horiba Penlra XLR hematology analyzer that utilizes Double Hydrodynamic Sequential System Principle.
MediumHanks balanced salt solution con
taining 7g of NaCI, 350 rug of N.HC02,350 mg of KCL. 200 rngof MgS04 • 7H20.55 mg of Na2HP04 • 2H20. and 55 mg ofKH2P03 per liter without addition of theusual amoum (0.09 %) ofglucose at pH 7.4.
and incubated for 30 olin at 370C undercontinuous agitation to opsonize the bacteria with antibodies from the serum sample.
The "coaled" bacteria \\1.15 cemrifuged and resuspended in NSS. II was thenwashed three times with NSS and suspended in Hanks balanced solt solution (HBSS)and adjusted to a final bacterial concemration of lS'Vfl iransmittance UJ a wavelengihof 420 nm, (Ordeonez et al., 2008)
Preparutlon of Bacterial SuspensionJriIjlIlJ'/ococcus auwfls pure culture
was purchased in UP Diliman Departmentof Microbiology. subcultured at 370C inBlood Agar Plate and harvested during exponcnrial growth. A I: I0 ratio of pooledSerum to Bacterial suspension \\'35 prepared
111is procedure for Phagocytic Activit)' has been reviewed and evaluated byUERM's chief pathologist. Dr. Araceli P.Jacoba, MD, FPSP.
rlu:,£oc'}'lil: Actj\'ity-r!usucylk ""• Phll!>OC)"'C Index
P~lndex....'Xo.oCSupby!1lo:0«i in.PhaECK""'C Nculropl!ilJ
Totll' Ni),urNC'Il11Upll-ib Oblll'f\"t"O
·~Jl"Ylk N.:uttopbib - Pt.lN5 dial phagOC)'lilC'd IIIIns, '~.:t1Ipblocooci
Phol£oeytk pM."en'...No.oC phagocyuc Nt!Ulmpbfls·TOlld No,orN,:ulfOpbib Oblcm'ed
Blood smears were also prepared from theheparinized blood samples prior to bacterialinoculation to serve as 'check slides'.
These slides were viewed to inspect for presence of toxic granulations.The Phagocytic Index was recorded as themean number of bacterin in the fitsl 40 neutrophils viewed under the microscope whilethe Phagocytic Percentage is the percentageof PMNs that has participated in ingestionof more than three staphylocci. PhagocyticActivity, which serves as the overall pictureof Neutrophilic Phagocytosis, is then cornpuroo by finding the product or PhagocyticIndex and Phagocytic Percentage. Parameters for phagocytosis are explained by thefollowing formulas. (Helium. 1977)
Effedl of l.f..Hotlr Fastitlg oa the YiI,-oPh.agocrtir :\(tiYtly or NflltropbUs
A Pearson product-moment correlation coefficient {PPMCC} \\'3S computed to assess the relationship between the change in WBC Count and the change in PhagocyticActivity. Results in Table 3 show tl,at change in WBC is not correlated with change in
.0005.034'1.66 19 3~', . 192"U3
7.0S0.894.80PhegocyticActh'iI)''p<.05.
KI SD sf SD dJ SiS
Before Aft('r 95% CI fOrFasting Fasting Mean Diiferma:
Table 2/lIlSU/tSolY.f~·/ .'II/{IIk,,"'Cnptil-'t! Star/stfty!br F.1,{'ti/1S;l/,d Pb.~¥oc;l'fii:AC/li'llj;
Since both Phagocytic Index and Phagocytic Percentage have shown significant increase after the fasting period, it naturally followed thai Phagocytic activity, the computedvalue derived from the product of both of the fore-mentioned parameters, \\115 also shown10have significantly increased at the end of the 24-hour fasting period.
Results in Table 2 show a statistically significant difference in mean phagocytic activity before and after fasting. Mean Phagocytic activity was reported to be4.80±O.89 and 7.08:>1.66before and after fasting, respectively (p<O.05). This constitutes an average of 5414% increase on the overall activity of Neutrophil, after the fasting period.
Fasting F~ing Mean DiJftrC'n«~vf SO ~I SD df r SiS
PbagG- 6.12 0.90 8.16 1.70 19 3.13, .11S' 4371' _000C)1ic )_'1IndexPbag.. 0.78 0.06 0.87 0.05 19 3.02, .0;4' 4539' .000cytic 1.06pcrcC'nl3gc'p<.05.
Table IDc!~'l'jp(ivi' .s1<1lJ:\·(ic:.~and J:1~Y JlC'",~ullslor PhaGOC)vc: 01<1<>%.1.11dPh,woc)'IiC PC«'c'l]rJGc'
Before Afttr 9>~a for
between the samples collected and processed before and after fasting. Statistically significant changes were seen both in phagocytic index: r- .4.371. p<O.05) and phagocyticpercent t= 4.539, (p<O.05) as displayed in Table I.The mean phagocytic index beforefasting was 6. I2±O.90 and 8.16±1.70, (p<O.05) after the fasting period, Additionally, thelucan phagocytic percent "vas 78o/"!:6°1nand 870/0:1:5%(p<O.OOO) before and after fasting.respectively.
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,.--~.," ."1: ~
Figure 2.1 ScatterPiOts"';j,";Sentlllg betore and alter tasung va'i'UeS'";;:phagocyticIndex (y
.... .'.:.- ....
:
III
Figure 2.1 shows scatter plots that graphically illustrate a strong positive correlationbetween phagocytic index and phagocytic activity while Figure 2.2 illustrates a weak positive correlation between the phagocytic percentage and phagocytic activity. This furthersupports thal the change in phagocytic activity is mainly due to the phagocytic index ratherthan the phagocytic percent.g •.
riillln"/' BaTgraph showing the percent increase in parameters on phagocytosis before andafter fasting period.
• non·fasting.f;tJO>ing
Figure I illustrates the percent increase in all of the parameters for phagocytosis. It can benoted that the increase in Phagocytic Activity was principally due to the increase in thePhagocytic Index rather than the Incr .. ", in the Phagocytic Percentage .
Statistical analyses in Table 3 provided no evidence that the number of WBe. isrelated to the phagocytic activity, This would lead to the conclusion that an increase inphagocytic index is associated with enhanced function rather than the decrease of numberof leukocytes,
.128 NS
r Sig VI
·.J52Change in \\fBC Count
Phagocytic Activity. r (20) = -.352. P =.128.TabidCorn>/at/oll ol'C'j)t)IJG~s/1) flfBC COllill ;'iJJdP/J/l_§oc.ytl,· Actillity
Chanp, in Ph.lOOQ1kActJritr
Effedl of l.f..Hotlr Fastitlg oa the YiI,-oPh.agocrtir :\ctiYtly or NflltropbUs
Ot'Sl,;ripll;'l-' Stllfisties ,711U7=t(;',)(,k,..,y/ts lor Blood COIlnlBeron: After Sigel forFilSllng Fn.~tiog ~ieaN Difference
95~..M SD M SD df T Sig,
RBC 5.0-J 0.37 5.20 OA3 is .13. .935- 4.640· .000.()Q
llGB 1-l.75 1.53 15.11 1.61 19 .65, .968' 5.081- .000.17
llCT 44..11 3.99 45.75 424 t9 1.95. .953- 4.654- .000.74
~ICV 88.16 5.49 88.2t 5.46 t9 .19. .996 0.461 .6~_.10
~ICH 28.78 1.13 29.01 lAS t9 1.45. .429 0.511 .608·.87
~IC- 31.72 2.33 32.01 1.18 t9 1.43. -.041 ·.130 .898HC -1.62ROW 11.35 1.23 12.25 1.17 t9 .07. .Q54 -1.214 .240
·27PlT 351.05 53.64 .144,68 62.34 t9 .9.95. .1l10 ·.817 .424
12.68rvlPV 8.0 0.49 S.J5 0.57 t9 J.I. .750 1.815 .085
U.iHnilYR_"-h J... r...1 \'01,,11' III :-.-.. 1
Table 4 shows the summary of Complete Blood Count values of all participants.These values were within the laboratory normal range for both male and females duringeach oCtlle time periods in which bleed was drawn. Funhermore, it also exhibits signitical\ttrends in eBe parameters following the 24-hour fasting period.
Analysis of subjects' eBe showed that Total WBe count, RBC. Hemoglobin andHematocrit were significantly increased at 0.05 significance level. Percentage and absolutecounts of Neutrophils were also increased at the end of the fasting period while LY1UpJtoeyre, Monocyte find EosillOphii percentages and absolute coums have all signific.alllly decreased. Other sHltislical results did tlot have significant difterences.Table 4
Flgurel.l Scatter plots representing before and after fasting values ofphagocytic percentage (y axis) versus phagocytic activity (x a:xis).
!-If --
Li
axis) versus phagocytic activity (x axis).
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The above results for index of and percent phagocytosis, for example, demonstratethai roughly 480 staphylococcus would be phagocytised by 100 PMNs before fasling.while after fasting. this number would reach 708. Therefore. it seems that after fasting.the immune system may respond more actively to infection (by gmm positive bacteria. forexample) than before fasting,
Since Q signiticam increase in \VBe count was observed before and after fasting.PPMC 'vas performed to establish that the increase in WBC was not the cause of the increase in phagocytic activity, The result showed no correlation between the increase in\VBC count and phagocyticactivity, therefore the increase in phagocytic activity was due to
In this study, the function of neutrophils was evaluated based on three parameters:the phagocytic percentage, which shows how many of the observed neutrophils are able toundergo phagocytosis; pbagocytic index which reflects the ability of neutrophils to engulrbacteria and phagocytic activity which determines the overall function of the neutrophils,Results showed a significaJu increase IIIall three parameters after a 24-l1oul' fast 111esigniticafll increase in Phagocytic Activity following t11C14--hour fasting period establishesthai the-re is a relationship between a 24-hour fast and Neutrophilic Phagocytic Activity.Increase in these parametersmay have protective effects on the responses to infection andother pathogenicity.
The innate immune system, which includes phagocytic cells. tonus (he first line ofdefense against microbial disease. especially extracellular pathogens. Neutrophils or polymorphonuclear cells (PMN) funclion as phagocyte by following chemotactic cues 10 locatesites of inftrunnunion or infection and removing ure injurious egem through engutfmem &digestion. The role of neutropbils as primary responders during bacterial infection is crucial. If sI0\\'5 [he spread of infection and allows the immune cells to mount a more specificresponse towards the pathogen. (Czerkinsky & Holmgren. 2005)
Effedl of l.f..Hotlr Fastitlg oa the YiI,-oPh.agocrtir :\(tiYtly or NflltropbUs
-.02woe 8_43 1.54 9.38 2.01 I· 1.67. .653- !.769~ .012
23NEU 54.48 7.&3 61.48 6.73 19 10.33. .531- 4.39&- .000
3.67LYM 3l.78 5.76 28.70 5.60 19 .2.6-1. .587- 4J6S· .000
-7.52MON 1A3 1.76 6Al 1.17 19 -A2. .70.1* -3.602* .(X)!
·1.59EOS 3.n 1.97 2.78 1.17 19 ·.06. .390' ·2.24Q· .037
·1.81BAS 0.61 0.15 0.6l 0.11> 19 .12. .35t 0.43l .670
·.08"p<_o.s.
IV. DISCUSSION
After 24-30 hrs, liver glycogenstores are mostly depleted and glucoselevels drop and maintained at 75-80 mglL(Warade. 2014). This marginal decrease inglucose level after 24-bour fasting can beimplicated to cause a positive increase inphagocytic activity where lowering of bloodglucose levels of diabetic patients or experimental animals has been reported to havesignifi(''anl correlation \vith hnprovememof neutrophil functional activity (Van Oss,1971)
A very recent study conducted inihe University of California (publishedJune of2014) has shown intermiuent, prolonged fasting induces changes thnt triggerstem cell-based regeneration of new immune system cells, In particular, "prolongedfasting reduced the enzyme PKA, shiftingstein cells from a dormant state to a stateof self- rencwal". In mice, il was describedthat fasung cycles '"flipped a regenerativeswhch," Changing the signaling pamwaysfor hematopoietic stein cells. which are responsible for the generation of blood andimmune systems. (Chia-Wei et al, 2014)These findings suppon this study's data tbrurevealed a significaut increase ill cae pol-
hanced phagocytic activity is enhancedchemotaxis or movement of phagocytes towards the antigen. In 3 study on two groupsof male and female mice in fasting conditions. an increased immunoglobulin levelsin colon nlUCUS and cortisol. interleukin 10(LL-IO). 1l11d inrcrferon gamma (rFN-"r) illplasma were shown. (Hiramoto et, AI, 2008)Treatment of_PMNs \\'ith IFN-y was demonstratcd to have significant effects on signaltransduction, gene expression, functions ofphagocytosis and cell killing. (Shalaby etal., 1985;Wnlker& Wnrd, 1992)
Another possible reason for en-
The increase in phagocytic activitymight be due to several mechanisms thatwere described by earlier studies. This includes 3D increase in the titer of opsonins,enhanced chemotaxis. decreased glycemiclevel and generation of newer, more viablewhite blood cells. In a study that was conducled by Khahazaei on the effect of fastingon the immune system of athletes duringRamadan, Ihey have observed that fastingseems to have positive effects 011 increasing the serum levels of C4, IgA levels. C4,a complement protein, can be cleaved intoC4n and C4b. of which C4b is no opsonin.An opsonin is a molecule that coats targetantigen and serves as signals for phagocytesto engulf IgA on the other hand is all antibody that plays a critical role in mucosalinununity that prevents infectious diseasesin gut and lung tissues. Titerof opsonins andnumber of opsonized bacteria are variablesthat are directly proportional to Phagocytic Activity. (Gentile, Conte & Formisano,2004)
enhanced function of the neutrophils. Scatterplots correlating phagocytic activity nodphagocytic index and phagocytic activityand phagocytic percentage was prepared 10show whether phagocytic index or percentage positively influenced the rise in phagocytic activity, It showed that the increase inphagocytic index or the ability of individualneutrophil 10 engulf bacteria was a majorfactor in the increase in phagocytic activity.The number of neutrophils that were able tophagocytose nl least 3 bacteria as shown bythe phagocytic percentage also increased by11.4% but is 110l sltOllgly correlated (0 tbeincrease in phagocytic activity,
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Belief and the Healing Arts of AncientCivilization. World Research Foundation, Retrieved fromhttp://\V\\'\v.\vrf.org/acient-med icine/belief-and-thc-healing-arts-of-ancient-civilizations.php. January IS, 2014.
Brown LK .•& Mussell K. (1984). Ethnicand Regional Foedways in theUnited States: The Performance ofGroup Identity. Knoxville,TN: University of Tennessee Press
Alba-Loureiro T.C.. Munhoz C.D.. MartinsLO;Cerchiaro. G.A.. Scavone C..Curi, R. & Sannomiya, P.(2007)Neutrophil Function and Metabolism in lndividuals with DiabetesMellitus. Brazilian loumal of Medical and Biological Research.Aug;40: 1037-1044.
Alba-Loureiro I.e.. Hirabara S.M..Mendonca J.R.. Curi R., & Pithon-Curi T.C. (2006) Diabetescauses marked changes in functionand metabolism of rat neutrophils. Institute of biomedicalSciences, University of Sao Paulo.188: 295-303. Re .trieved from hrtp:l/"",·\v.ncbLnlm.nih.gov/pubmedJ 16461555
Aadil, N.. Houti, L E.,& Moussamih,S. (2004). Drug intake during Ramadan, BMl: British MedicalJournal (International Edition).329(7469),778.782.
The design of future studies thatwould employ the use of more analytic testmethodswhlch makesuse of'flow-cytomerric test SYStClllS that simultaneously measures phagocytosis and production of reactive oxygen species (ROS) ofneutrophils isrecommended(0 confirm and to naveamoreaccurate reading of the parameters that havebeen measured in this study. This would alsogive the future researchers a more completeset of data that would describe neutrophilactivity under fasting conditions.The researchers also recommend a follow-up study regarding the duration of thebeneficial effecrs all t11Cneuuephil phagocytic activity after Ole 24--hour fasting period.
VI. RECOMMENDATIONS
This study \V3S intended to determine if there is :1 relationship between 324-hour religious fasting on neutrophilicphagocytosis. The study found that 24·hourfaslillg significantly enhanced the I1('UlfO
phils capacity to engulf'bacteria, which maybe an ifuportant beneficial effect of religiousfasting. These observations become meaningful when it is recognized that phagocytesis is the rate limiting step in the reductionof viable organisms. Thus. diet rna)' playakey role in Olecontrol of resistance to infection.
V.CONCLUSION
rameters like RBC count. Hemoglobin. Hematocrit, \\'Be count including the relativecount for .11 5 WBC types - neutrophils,lymphocytes, monocyres. basophils and eosinophils.
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