ACTA FAC MED NAISS UDC 613.287.1-074:616-053.2

SUMMARY

Human milk is the main source of nutrients for a child during the firstmonths of life. Alkaline phosphatase (EC 3.1.3.1) is a very important enzyme inclinical chemistry because of its activity in various tissues and biological fluids,being an indicator of physiological or diseased states. Milk contains severalphosphatases, the principal ones being alkaline and acid phosphomonoesteraseswhich have no known function or significance in milk, and have been studiedextensively so far.

The objective of the present study was to determine the alkalinephosphatase activity and concentration of inorganic phosphorus (P) incolostrum and maturemilk obtained from nursing mothers.

Our longitudinal biochemical analysis showed that alkaline phospha-tase activities decreased from the 1 and the 2 day of lactation (colostrum) tothe nd of the first month of lactation (at day 30). The amount of inorganicphosphates from colostrum increased to the end of the first month (at day 30) oflactation. The alteration between colostral and mature milk alkaline phospha-tase activity may be a consequence of theALPtransfer from the blood of motherinto the colostrum and milk during breastfeeding.

The concentration of nutrients in colostrum and mature milk suffersalterations, including a decrease in alkaline phosphatase activity and anincreasein P, probably in order to satisfy the requirementsof the nursing infant.

: human milk, colostrum, alkaline phosphatase, inorganicphosphor

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Ljiljana Bjelakovic , Gordana Kocic ,Tatjana Cvetkovic , Dusica Stojanovic ,Stevo Najman , Zoran Pop-Trajkovic ,Marina Jonovic , Bojko Bjelakovic

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Corresponding author. Ljiljana Bjelakovic • Tel: +38118534579 • E-mail: ljilja975@gmail.com

ALKALINE PHOSPHATASE ACTIVITYIN HUMAN MILK DURINGTHE FIRST MONTH OF LACTATION

Original article

ACTA FAC MED NAISS 2009; 26 (1): 43-47

INTRODUCTION

Human milk is the main source of nutrientsfor a child during the first months of life. Milksynthesis starts in the epithelial cells of the mammarygland at the end of pregnancy to support the nutritionand promote the health of newborn infants. Milkcontains vital nutrients such as proteins, carbo-hydrates, lipids, minerals and vitamins, together withbioactive substances including immunoglobulins,hormones and enzymes (1-3). Over 70 indigenousenzymes have been identified in the milk of various

mammalian species (4,5). Some milk enzymes areconstitutive components of milk, while others areinduced at particular periods of the lactation cycleand some pathological conditions. The enzymesystems normally present in mother's milk mayinfluence the health and nutrition of the newborninfant. The occurrence of a phosphatase in milk wasfirst recognized in 1925 by F. Demuth (6).Subsequently, ALP was characterized as an indige-nous in milk (7-9).

Milk contains several phosphatases, theprincipal ones being alkaline and acid phospho-

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Pediatric Clinic, Clinical Center Nis, Serbia,Institute of Biochemistry,

Faculty of Medicine in Nis, Serbia,Department of Hygiene and Epidemiology,

Public Health Institute Nis, Serbia,Institute of Biology and Human Genetics,

Faculty of Medicine in NisClinic of Obstetrics,

Clinical Center Nis, SerbiaGynecology and

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Ljiljana Bjelakovic, Gordana Kocic, Tatjana Cvetkovic, Dusica Stojanovic, Stevo Najman, Zoran Pop-Trajkovic, Marina Jonovic, Bojko Bjelakovic

The samples of human milk were obtainedfrom twenty healthy mothers of term infants at days1,2 (colostrums) and 30 of lactation.

The milk sample was obtained fresh, afterthe infant had sucked for 5 minutes with a manualbreast pump (Ginevri, Milan, Italy Samples ofhuman milk (n = 90 were stored at -20°C untilanalyzed.

Alkaline phosphatase (EC 3.1.3.1) activitywas measured at pH 10.3 using p-nitrophenylphosphate as substrate by using commercial Elitech-France kit, ALP-DEA. The concentration of inorganic phosphate (P) was measured with commercial kitElitech-France on the basis of the formationphosphomolibdate (12, 13). The estimated sampleswere dissolved ten times before applying to thebiochemical analyzer.

STATISTICS

Descriptive data were presented as a meanvalues with a standard deviation. The obtainedresults were statistically analyzed using one wayANOVA trend analysis with SPSS computer statistical program. The value p<0.05 was considered statistically significant. All statistics were done usingthe SPSS computer package version 13 (SPSS,Chicago, IL, USA).

RESULTS AND DISCUSSION

The analysis for alkaline phosphatase (ALP)and inorganic phosphate (P) in human colostrumsand milk are presented in Graphs 1 and 2 .

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monoesterases which have no known function orsignificance in milk and have been studied extensively so far ( 10,11 ).

Alkaline phosphatase (AlP) (EC 3.1.3.1) is amembrane-bound glycoprotein that is widelydistributed in human tissues. There are four principaltypes of humanALP: intestinal, placental, germ-cell,mammary tissue and bone/kidney/liver (tissue non-specific). The intestine and placenta are particularlyrich sources and ALP. There are slight differencesbetween the ALPs in the bone/kidney/liver and othertissues, probably including mammary tissue, mainlyin the degree of glycosylation. The gene for humanbone/kidney/liver (tissue non-specific) ALP is atleast five times larger than that for intestinal ALP(12).

Alkaline phosphate (EC 3.1.3.1) is a veryimportant enzyme in clinical chemistry, since itsactivity varies in different tissues and serves as aspecific indicator of diseased states, especially ofbones and liver. However, in spite of the intensiveresearch on alkaline phosphates and its widespreaddistribution, its physiological roles are not yet known(13,14). The aim of the present study was todetermine the alkaline phosphataseALP(EC 3.1.3.1)activity and concentration of inorganic phosphorus(P) in colostrums and mature milk obtained fromnursing mothers.

MATERIAL AND METHODS

This longitudinal study involved 30 healthywomen admitted to the Clinic of Obstetrics andGynecology in Clinical Center Nis, Serbia, fordelivery betweenOctober and December 2008.

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Alkaline phosphatase activity in human milk during the first month of lactation

activity, which may suggest a role of these cells inmilk secretion (16,17). ALP can exist in blood in thecomplex with protein, as enzyme- immunoglobulincomplex as well as ALP-lipoproteins complex(14,18,19). It is well-known that colostrums areimportant sources of immunoglobulins and lipids(20)

Colostrum milk is one of the most importantfoods a newborn can receive from his mother soonafter his birth. When the milk produced in themother's body changes from colostrum milk to nor-mal milk, the level of total protein, fat, total solidsdecrease, lactose increases and colostral Ig declinesrapidly. The decreasing of ALP in our milk samplesmay be explained according to decreasing of imuno-globulins and lipids during the lactation (21,22).

ALP act on a large variety of naturallyoccurring substrates, but the natural substrates onwhich the enzyme acts in the body are not known.ALP can dephosphorylate casein, phosphoprotein,under suitable conditions (23). As a phosphomonoesterase ALP may act on monophosphate esters ofcarbohydrates or lipids like glucoso-6-phosphate,galactoso-6-phosphate or 3-phosphoglicerin aldehyde, dioxyacetone phosphate, diacylglicerol phosphate or complex lipids, phosphatydilethanolamine(12). Based on these data we expected that the con-centration of inorganic phosphate would follow theALP activity. However, we obtained the oppositeresults.

The concentrationof micronutrients in colostrum and mature milk suffersalterations, including adecrease in Fe, Zn, K and Na and an increase in Caand P, probably in order to satisfy the requirementsof

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Our longitudinal biochemical analysisshowed that alkaline phosphatase activitiesdecreased from 166.66 ±11.563 Units/L at day 1 oflactation and 173.478± 12.462 U/L at day 2(colostrums) to 78.125± 4.021 Units/L at the and ofthe first month of lactation (at day 30 ) p<0.05.

Inorganic phosphates increased linearlyfrom 1.242 ± 0.049 at the end of day 1 of lactation and2.177± 0.110 mmol/Lat day 2 (colostrums) to 2.336±0.105 mmol/Lat day 30 of lactation.

DISCUSSION

About 70 inherited enzymes have beendiscovered in milk (5,6,8). Those enzymes which arepresent at the highest levels have been isolated frommilk and characterized. Among them are thenaturally present enzymes of serum, like transaminases (ALT and AST), phosphatases (alkaline andacid), lactate dehydrogenase (LDH), creatinphosphokinase

(CPK, -amylase, aldolase, ribonucleases, -glutamyl transferase, lysosyme- lactenins, catalase,xanthine oxidase, sulphydryl oxidase - SHOx, glutathione peroxidase, -acetylglucosaminidase- NAG,superoxide dismutase-SOD, and others (9). Becauseof that, enzymology of mammalian milk enzymes isvery intensive today.

There are a few recent researches on milkalkaline phosphatase.

The inherited ALP in milk is similar to theenzyme in mammary tissue (14, 15). Most of theALPs in the mammary gland is in the mycoepithelialcells; the epithelial secretory cells have low ALP

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the nursing infant (24 - 26). Our results, concerningthe decrease of inorganic phosphates levels in milksamples, are in agreement with these data and withthe observation that ALP is inhibited by inorganicphosphate (24).

The ALP activity of bovine milk variesconsiderably between individuals and throughoutlactation, minimum at week 1 and maximum atweek 28 (26).

Our results considering high alkalinephosphatase activity in colostrum, during the firstand second day and lower enzyme activity at day 30of lactation, are in agreement with the that,in general, the enzyme content of human colostrum ishigher from that in the corresponding mature milk(21,22). The alteration between colostral and maturemilk alkaline phosphatase activity may be theconsequence of the ALP transfer from the blood ofmother into the colostrum and milk duringbreastfeeding.

Namely, it is well-documented that duringgravidity, the ALP in mother’ blood increasesproportionally to the augmentation of the mass of theplacenta (12 -14).

Some data show that increased dietary fatintake in lactating women increased the levels oflipase, esterase and alkaline phosphatase (17,18)enzymes that play a role in digestion and metabolismof fat; at the some time the protein supplementationof malnourished women increased alkaline phospha-tase activity in their milk (27, 28 ).

Considering the origin of milk enzymes,e.g., from mammary cells or blood, their activities in

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milk are not constant, but vary significantly due todifferent physiological and health states of thelactating mothers: due to the stage of lactation, diet,stress, mastitis or other factors (10). Among the mostsignificant features of milk enzymes are those usedas indices of mother’s and baby’s health.

CONCLUSION

Alkaline phosphatase (EC 3.1.3.1) is a veryimportant enzyme in clinical chemistry because of itsactivity in different tissues and biological fluids,serving as an indicator of physiological functions ordisease states. Milk contains several phosphatases,the principal ones being alkaline and acidphosphomonoesterases. They do not have precisefunction or role in milk and until to date have beenstudied extensively. The objective of the presentstudy was to determine the alkaline phosphataseactivity and its concentrations of inorganicphosphorus (P) in colostrums and mature milkobtained from nursing mothers. Our longitudinalbiochemical analysis showed that alkaline phospha-tase activities decreased from the 1 and the 2 day oflactation (colostrum) to the end of the first month oflactation (day 30). The amount of inorganic phospha-tes increased from colostrums to the end of the firstmonth (day 30) of lactation.

The alkaline phosphate activity which isnormally present in mother's milk may influence thehealth and nutrition of the newborn infant.

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REFERENCES

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Pavlovic D, Kocic G. Biohemija. Savremena Administracija,Beograd. 2006:1018-1026.

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7. Morton RK. Alkaline Phosphatase of Milk. 2.Purification of the enzyme Biochem J 1953; 55:795-800.

tečnosti. Mleko.U knjizi: Koracevic

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9. Kelly AL, O'Flaherty F, Fox PF. Indigenousproteolytic enzymes in milk:Abrief overview of the present stateof knowledge. Inter Dairy J l 2006;16: 563–572.

10. Kelly AL, Fox PF. Indigenous enzymes in milk: Asynopsis of future research requirements. Intern Dairy Journal2006;16: 707–715.

11. Silanikove N, Merin U, Leitner G. Physiologicalrole of indigenous milk enzymes: An overview of an evolvingpicture International Dairy 2006:533–545.

12. Moss DW, Henderson AR, Kachmar JF. Enzymes.In Tietz NW (Ed), The textbook of Clinical Chemistry.W.B.Saunders Company 1986: 619-774.

13. Werner CW, Lott AJ. Alkaline phosphatase. In:Kaplan AL and Pesce AJ (ed). Clinical Chemistry: theory,analysis and correlation. 1984:1094-1098. The C.V. MosbzCompany.

14. Koracevic D. Enzimi. U knjizi : Koracevic D,Bjelakovic G, Djordjevic V, Nikolic J, Pavlovic D, Kocic G .Biohemija. SavremenaAdministracija, Beograd. 2006:1-162.

15. Morton RK. Alkaline phosphatase of milk. 1.Association of the enzyme with a particulate lipoproteincomplex. Biochem J 1953; 55(5): 786–795.

Ljiljana Bjelakovic, Gordana Kocic, Tatjana Cvetkovic, Dusica Stojanovic, Stevo Najman, Zoran Pop-Trajkovic, Marina Jonovic, Bojko Bjelakovic

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AKTIVNOST ALKALNE FOSFATAZE U HUMANOM MLEKU U TOKU PRVOGMESECA LAKTACIJE

SAŽETAK

Humano mleko je glavni izvor nutritivnih sastojaka za decu u toku prvog meseca života.

-ka (prvi i drugi dan laktacije

ostruma i pravog mleka mogu da budu posledicaprelaskaALPiz krvi majke u toku dojenja u kolostrum ili mleko.

Koncentracija hranljivih sastojaka u kolostrumu i pravom mleku trpi promene, verovatno prema

potrebama bebe koja doji.

: humano mleko, kolostrum, alkalna fosfataza , neorganski fosfor

Ljiljana Bjelakovi , , , ,Stevo Najman , ,

, i entar Niš, Medicinski fakultet Niš, SrbijaBiohemijski nstitut, Medicinski fakultet Niš, Srbija

Institut za igijenu i pidemiologiju , Medicinski fakultet Niš, SrbijaInstitut za iologiju i umanu enetiku, Medicinski fakultet Niš, Srbija

Klinika za bstetriciju inekologiju i entar Niš,Medicinski fakultet Niš, Srbija

ć Gordana Kocić Tatjana Cvetković Dušica StojanovićZoran Pop-Trajković , Marina Jonović Bojko Bjelaković

Klinika za dečije interne bolesti Kliničk ci

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o i g , Kliničk c Srbija

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Alkalna fosfataza ( EC 3.1.3.1) je veoma značajan enzim u kliničkoj hemiji jer je njenaaktivnost u raznim tkivima i biološkim tečnostima indikator fizioloških i patoloških stanja.Mleko sadrži nekoliko fosfataza, od kojih su najznačajnije alkalna i kisela fosfomonoesteraza,čija je funkcija još uvek nejasna i pored intenzivnog izučavanja. Predmet prikazane studije biloje ispitivanje aktivnosti alkalne fosfataze (ALP), kao i neorganskog fosfora u kolostrumu imleku koje je dobijeno od majki dojilja. Naša longitudinalna ispitivanja su pokazala da seaktivnost ALP smanjivala počev od prvog i drugog dana laktacije (kolostrum) do kraja prvogmeseca laktacije (30. dan). Koncentracija neorganskog fosfora se povećala, počev od kolostralnog mle ) do kraja prvog meseca (30. dan laktacije). Promene uaktivnosti alkalne fosfataze između kol

,ukljućujući smanjenje aktivnosti ALP i povećanje neorganskog fosfora

Ključne reči

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17. Hamilton TA, Górnicki SZ, Sussman HH.Alkaline phosphates from human milk. Comparison withisoenzymes from placenta and liver. Biochem J 1979;177:197–201.

18. Karmarkar MG, Rajalakshni R, Ramakrishnan CV. II.Activities of Certain Milk Enzymes in Relation toDietary Fat intake.Acta Paediatrica 2008;52:554 – 556.

19. Karmarkar MG, Ramakrishnan CV. Relationbetween Dietary Fat, Fat Content of Milk and Concentrationof Certain Enzymes in Human Milk. J Nutr1959;69: 274-276.

20. Csapo-Kiss Z, Stefler J, Martin TG, MakrayS, Csapo J. Composition of Mares' Colostrum and Milk.Protein Content, Amino Acid Composition and Contents ofMacro- and Micro-elements. International Dairy Journal1995; 5:403-415.

21. Hadorn U, Hammon H, Bruckmaier RM,Blum JW. Delaying Colostrum Intake by One Day HasImportant Effects on Metabolic Traits and onGastrointestinal and Metabolic Hormones in NeonatalCalves. J Nutr 1997;127:2011-2023.

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22. Maden M, Birdane FM, Altunok V, Dere S. Serumand colostrum /milk alkaline phosphatase activities in thedetermination. 2004;11: 565-569.

23. Jasinska BK, Kleczkowski K, Michalak W.Influence of -Lactoglobulin on Milk Alkaline PhosphataseActivity Toward the Main Milk Caseins. J Dairy Sci1985;68:2172-2175.

24. Mastroeni SSBS, Okada IA, Rondó PHC, DuranMC, Paiva AA, Neto JM. Concentrations of Fe, K, Na, Ca, P, Znand Mg in Maternal Colostrum and Mature Milk. J TropPediatrics 2006 52(4):272-275.

25. Lucas A, Brooke G, Baker BA, Bishop N, MorleyR. High alkaline phosphatase activity and growth in pretermneonates.Archives of Disease in Childhood 1989; 64: 902-909.

26. Nagra SA. Longitudinal Study in BiochemicalComposition of Human Milk During First Year of Lactation. JTropical Pediatrics 1989; 35 :126 -129.

27. Hibberd CM, Brooke G, Carter ND, Haug M,Harzer G. Variation in the composition of breast milk during thefirst 5 weeks of lactation: implications for the feeding of preterminfants. 1982;57: 658-662 .

28. Boss MH, Batt WG. Alkaline phosphatase activityand diet. J Nutr 1956: 137-144.

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Alkaline phosphatase activity in human milk during the first month of lactation