I ro. J. Med. Biochem., 2001, 5(1&21

Study on MDA and Urinary Albumin Excretion in NIDDM Patients

Subhashree Ray, Srikrushna Mohapatra, Pramil la K. Mishra and Priyamohan Mohanty

Department of Biochemistry, M,K.C.G. Medical Col lege,Berhampur, Orissa

AbstractFree radical status (as MDA) and urinary albumin excretion (UAEI were studied in 55 NIDDM malepatients along with the 20 age and sex matched controls, The results revealed a correlation

between fasting blood sugar and rise in MDA in patients with microalbuminuria {r= 0.541}. There

was also a posi t ive correlat ion between r ise in MDA and UAE lr=O.7421.

lntroductionDiabet ic nephropathy is def ined as the onset ofpersisterrt proteinur ia with raised blood pressurein pat ients with chronic diabetes mel l i tust. Thel i fe expectancy in such pat ients is reduced withhigh r isk of renal f ai lure or cardiovasculardisease, ,or both2. The condit ion of incipientdiabet ic nephropathy is character ised bypersistent microalbuminuria3 in which there is analbumin excret ion at a higher rate than normal

but below than those giving posit ive results on

dipst ick test inga. Microalbuminuria is consideredto be present when the ur inary albumin excret ion(UAEI is greater than 2Opg/min and less than orequal to 2OO pg/min. This level corresponds toapproximately 3O-3OO mg/24hrs5.

The rolo ol f ree radicals on oxidat ive stress in

diabetes has also gained much importance

recent ly6. with this end in view, f ree redical

status and UAE were studied in 55 NIDDMpatients to evaluate the relat ion between l ip idperoxidat ion with microalbuniminuria and fast ing

blood sugar (FBSI.

Materials And MethodsA total of 55 known male NIDDM cases

between30-60 years of age were selected from

the diabet ic pat ients, at tended M.K.C.G. Medical

Col lege, Berhampur, Or issa. None of thesepat ients had overt renal impairment or

hypertension , nor any ret inal , cardiac or any

other organic disease. None of them were

smokers or alcohol ics. A total o l 20 heal thy age

and sex matched controls were also selected for

comparis ion. Selected NIDDM pat ients were

div ided into 2 groups. Twenty pat ients wi thout

microalbuminur ia having diabetes wi th a durat ionof 8.05 t 3.09 yrs belonged to group I and 35with microalbuminur ia who had diabetes for10.51 f 3.8 yrs to group l l . Fast ing blood sugarwas est imated by GOD-POD methodT. Urea and

creat in ine were est imated by Diacetyl Monoxime

&Jaffe 's method respect ivelys. Ur ine was

cof lected tor 24 hours, using 5N HCL as thepreservat ive and was assessed tor

microalbuminur ia by micral str ip test

manufactured by Boehr inger Mannheim Ltd.

Serum MDA was est imated by Thiobarbi tur ic

Acid react ions. Al l readings were taken in a

spectrophotometer, Systronics model 106.

Stat ist ical s igni f icance was done by ' t ' test and

correlat ion was calculated by ' r ' value.

Resul ts

Values foe blood urea , FBS, Serum creat in ine,

MDA and 24-hrs ur inary albumin excret ion for

NIDDM pat ients and heal thy controls are shown

in Table 1 . The resul ts revealed that the levels of

lnd. J. Med. Biochem.,2OO1,5(1&2)

FBS, MDA and UAE were most signi f icant ly highin pat ients with microalbuminuria. NIDDMpatients without microalbuminuria also showedhigher concentrat ions for FBS and MDA but thevalues wgre comparat ively lower.The correlat ion of FBS with MDA betweencontro, ls and NIDDM pat ients were done and theresults; ( ' fable 2) showed a. posi t ive correlat ion ofFBS to MDA with a ' r ' value of 0.541 in NIDDMpatients with microalbumanuria. There was also aposit iv 'e correlat ion l r=O.742!- between MDA andUAE ( ' fable 2) in pat ients with microalbuminuria.

DiscussionOxidat ive stress may bg a common pathway

l inking diverse mechanisms for the pathogenesis

of cormpl icat ion of diabetes mel l i tus6. Modif icat ionof long-l ived extracel lular proteins (e.g.

Crysl ;al l ins, Col lagen, Elast in, Myel in Sheathprot€l ins etc.) and structural changes in t issuesrich in these proteing (basement membranelcanlead to microangiopathy and nephropathy.Furtlrer oxidaitive stresE in the lipid of themenrbranes add to the impairment and damage ofcel lular membranes associated withathelrosclerosis and microangiopathy etc.6.In cl iabetes mel l i tus the mechanism of t ransi t ionfronr low to high levels of microalbuminuria is

unknown, but may be the result ofhaemodynamic; gbnormal i t ies, l ike increased

Healthy Controlsn-2O

2

transglomerular pressure gradientt0. l t has alsobeen suggested that microalbuminuria is aref lect ion of atherogenic vascular damagerr.I t has been demonstrated earl ier thatmicroalbuminuria may determine early renaldamage in type | ( IDDM) diabetesr2. We foundthat MDA concentrat ion was greater in NIDDMwith microalbuminuria compared to that ofpat ients without microalbuminuria, a simi larf inding has also been reported earl ierr3. Thepresent results showed a positive correlationlr =0.742l ' between MDA and UAE. Thiscorroborates with earlier reports of oxidativestress and basement membrane damage inNlDDM6. Since microalbuminuria is a potent ial

indicator of cardiovascular compl icat ion ofdiabetes mel l i tus as wel l as an early feature ofdiabet ic nephropathy, microalbuminuria couldresult f rom free radical damage. NIDDM pat ients

are exposed of f ree radical damage as evidenced

by increase in serum MDA. And the free radical

induced damage in renal basement membrane canlead to an increased UAE. Membraneperoxidation also exposes the vascular bed for

accelerated atherosclerosis and incidence of

hypertension. Thus, est imation of both MDA and

UAE, might be helpful in NIDDM pat ients to

evaluate free radical damage of membranes and

associated renal impairment as well as for better

management and appropriate therapy.

Table 1. Vrr lues for blood urea, FBS, Serum Creat inine, MDA and 24-hrs ur inary albumin excret ion(UAEI of NIDDM pat icnts and healthy controls.

Cases withoutMicroalbuminuria (Group - l)

n=20

Cases with Microalbuminuria(GrouP - l l l

n=35

Age { ln ,1 ' rs, f24hr U/-\E (mglFBS {nrg/dtlB. Urea (mS/dl)S. Crreatinlne (mg/dllS.MDA (mmol/ml l

43.45 r 8.7323.15 +4.1683a11.17

31.1O*3.581 .22 +O.301.11 r0.34

45.80 r 14.5827 .40 t 6.67

145 r 9.234.00 r 3.461 .28 * 0.401.92 ro.61'

48.69 + 13.83133.09 +49,99162.57 + 15.6837.O8 + 6.781 .30 +O.36

2.66 t O.69" 'b

Levels of signlfieance compared with controls: 'P(O.OO1

Levels of s igni f icaneg compared with group l : bP<O.O1

.nd. J, Med. Biochem..2OO1,5( l&21

Table 2. Correlat ion between fast ing blood sugar (FBS) and serum malondialdehyde (MDA) andbetween MDA and 24 hrs ur inary albumin excret ion (UAE) in NIDDM pat ients with or withoutmicroalbuminur ia.

Correlat ion between FBS and MDA Correlat ion between MDA and UAE

FBS MDA T UAE MDA T(mg/dl l (nmol/ml l (-g) (nmol/ml)

NIDDM without micro- 145.OOt9.20 1.92+O.61 O.424 27.4Ot6.67 1.92rO.61 -0.319albu minur ia (Gr. l ) (>o.o5l (<o.osl

NIDDM with microalbu- 162,57r15.68 2.66+0.69 0.541 133.O9*49.99 2.66+0.69 0.742rtr i r tut iu (Gr. l l ) l<o.oo1) ( <o.oo1)

Values within bracket indicate P value.

References1 . Anderson, A.R., Christiansen, J.S., Anderson, 8. Varley, H. Eds. (1988). Practical Clinical

J.K., Kreiner, S. and Deckert, T. (1983). Biochemistry, 6th edition CBS Publishers and

Diabetologia, lS : 496. Distr ibutors, New Delhi , p. 351.

2. Mogensen, C.E. (19841. ,t/. Engl. J. Med.,31O : 9. Satoh, Kei. (19781. Clinica Chemica Acta.,90 :,

356. 37.

3. Mogensen, C.E., Chr istensen, C.K., and 10. Hostet ter , T.H., Rennke, H.G., Brenner, B.M. et

Vi t t inghus, E. (19831. Diabetes,32 (Suppl 2 l t 64. a l . (19821. Am.J. Med.,72 : 375.

4. Miulruf Marrc, Gi l les Clratel l ier , Herve Lieblanc, 11. Patney, R.L. and Garg, R. (1999). Med. Update,9

Tam Tlran Guyene, Joel Menard and Phil ippe t 427,

Passa, (1 9881.8MJ,297 :1992.

12. Borch-JOhnsen, K., Andersen, P.K. and Deckett ,

5. Mogensen, C.E. (1 9871, Kidney lnt., 31 z 673. T. {19851. Diabetologia,20 : 590.

6' Bavnes' J'w' (1eelt' Diabetes' 40 :405' 13' J',,x; l; "til'l;,,1#;: ll"ll;rt;; '|

^.t';!;.'.,7. Varley, H. Eds. (19881. Practical Clinical 236 : 495.

Biochemistry, 6th edition CBS Publishers and

Distr ibutors, New Delhi , p.322.

lnd. J. Med. Biochem., 2OO1, 5(1&21

Serum lmmunoglobul ins in Nephrot ic Syndrome

Shashi Seth, Manju B. Pahwa, H. Lal and Harjeet Singh*Departments of Biochemistry and Pediatrics,

*Pt. B.D. Sharma PGIMS, Rohtak

Abstract

The serum immuglobulins along with some common blood parameters were studied in 20 cases ofnephrotic syndrome in the age groupof 5-15 years. Serum protein, albumin and A/G ratio weresignificantly decreased while total cholesterol and blood urea were increased in nephroticsyndrome as compared to that of controls. Among the immunoglobulins lgG was reducedsignificantly while lgA and lgM were not altered markedly. lt is interesting to note that thereduction in lgG was more pronounced in patients with total cholesterol > 5OOmg/dl but lgA orlgM didnot show any difference. The concentration of urinary protein and blood urea were alsosignificantly higher in patients with total

'cholesterol > SOOmg/dl compared to those who had

cholesterolconsistent with the incidence of relapse, response to immunosuppressive agents andetiopathogenesis of the disease.

lntroduct ionThe nephrot ic syndrome is character ised bypresence of proteinur ia, hypoalbuminemia,hypercholesterolemia, hyperl ip idemia and edema.The edema is a consequence of ei ther decreasedintravascular oncot ic pressure due to loss ofprotein or al terat ions in the basementmembraner. The loss of water and salt into theinterst i t ia l space leads to reduct ion in plasmavolume and that t r iggers the kidney to retainsodium.Shalhoub2 suggested that the syndromerepresents the cl in ical expressions of pr imaryimmunological abnormal i ty. Afterwards, a fewworkers reported altered levels of serumimmunoglobul ins in pat ients with nephrot icsyndrome but with conf l ict ing resultsr '3 'o.However, this communicat ion attempts toevaluate the serum immunoglobul ins in pat ientswith nephrot ic syndrome.

Mater ials And MethodsThe present study was conducted in thedepartments of Biochemistry and Paediatr ics. Agroup of 20 cl in ical ly diagnosed cases ofnephrot ic syndrome (15 males and 5 females) inthe age group of 1O-15 years were studied forlgG, ISA and ISM levels. The results werecompared with 20 age - matched healthyindividuals who served as controls. Al l the

hypoalbuminemia and hypercholesterolemia butdevoid of infect ious diseases, diabetes mel l i tus,acute and post infect ious nephri t is.Blood was collected from the patients beforestarting treatment and stored at -2oo c til lanalysed. Serum glucose was est imated byO-Toluidine methods, urea by diaacetylmonooxime (DAM) method6, protein by BiuretmethodT, albumin by dye binding method andA/G ratio was calculated. Serum cholesterol wasest imated by enzymatic ki tss, serum creat inine byalkal ine picrate6 and serum sodium and potassiumby f lame photometryro,Serum immunologlobul ins were measured bysingle radial immunodiffusion technique describedby Mancini et af '.

Results And DiscussionValues for the common blood biochemicalparameters and serum immunoglobul ins inpat ients with nephrot ic syndrome and thecontrols are shown in Table 1 . The nephrot icpat ient had signi f icant ly lower levels of selumprotein, albumin and A/G rat io compared to thatof controls. Serum total cholesterol and bloodurea levels were signi f icant ly higher. The resultsare consistent with nephrot ic syndrome. Thelevels of immunoglobul ins were reduced in thepateints, and among the immunoglobul ins lgGwas reduced signi f icant ly (p< O.OO1l. Fal l inserum in lgG those pat ients could be ei ther dueselected pat ients had proteinuria,

Ind. J. Med. Biochem., 2O01, 5(1&21

to increased ur inary loss or decreased synthesisor increased catabol ismt. In the early sevent ies i twas reported that serum immunoglobul in levelsare reduced due to their select ive loss throughdamaged glomerul i in nephrot ic syndrome3'aowing to their smal l molecular weight. Obviously,imrnunoglobul ins l ike lgG are removed at a higherrate than those with relatively higher molecularweight l ike lgM.Hypercholesterolemia is one of the signi f icantf indings in nephrot ic syndrome. And on the basisof total serum cholesterol, nephrotic patientswere divided into 2 groups, i .e. , pat ients whohad cholesterol >5OO mg/dl and <5OO mg/dl .Twelve patients had cholesterolBiochemical f indings and serum immunoglobul inswere compared between two groups and resultsare presented in Table 2. l t was found that theoat ients whose serum cholesterol levels weregreater than 5OO mg/dl showed comparat ivelyhigher blood urea and increased loss of protein intheir ur ine. The mean serum lgG concentrat ion inpatients with higher level of cholesterol was

Table 1. Common biochemical parameters

syndrome and controls.

5

nearly 5Oo/o of that with cholesterol <500 mg/dl .I t is interest ing to note that serum lgMconcentrat ion of the pat ients with cholesterol )SOOmg/dl was workers reported lower levels ofserum lgG and higher levels of lgM in nephrot icsyndromer2'r3. Shalhoub2 suggested that thisal terat ion in serum immunoglobul ins is attr ibutedto impairment of T- cel l dependent ant ibodyproduct ion. l t was reported that abnormal i ty inT-cel l funct ion could hinder the immune systemin producing lgG ant ibody to secondaryimmunogenic st imulat ion. l t seems probable thatnephrot ic syndrome could ini t iate this defect inimmune system, or conversely, inheri ted defectscould ini t iate nephrot ic syndrometo.ts. However,al terat ion in serum chemistry l ike high levels ofurea and cholesterol and low levels of totalprotein, albumin and A/G rat io as wel l asincreased loss of protein in ur ine associated withnephrot ic syndrome might have a directrelat ionship with the serum immunoglobul inspart icular ly lgG.

and serum immunoglobul ins in pat ients wi th nephrot ic

Control

n=20Patients

n=20

Serum Chemistry:Serum protein (g/dl)

Albumin (g/dl)

A/G ratioCreat inine (mg/dl)

Cholesterol (mg/dl)

Blood Urea (mg/dl)

Blood sugar (mg/dl)

Na. (meq/ l )

K'(nroq/ l l

lmmunoglobul ins (mg/dl l :lgGlgAigM

6.84 r O.1 23.68 +0.1 11 .18 rO.O41 .13 rO.O8

1 91 .3 r 6.7023.9t2.5275.5 r 3.3134.5 *4.14.03 r 0.1 I

152.2t10121 9.5 r 18.1210.3*,15.2

5.09 * O,20*2.18r0.16

0.80 ro.o8*1.O5+O.14

455.2+22.75*37.6 t 3.64*

67.5 r ,2.1133.1+4.34.60 r O.30

62.2 *.5 .9 * *

167.0 r .18.2198.8 +.21.9

Values are mean t SEM

Levels of s igni f icance compared to controls (Student 's t - test) : P*<O.O5; **P<O.OO1

Ind. J. Med. Biochem., 2O01, 5(1&2)

Table 2. Comparison of Serum Chemistry and immunoglobul insserum cholesterol .

6

in nephrot ic pat ients on the basis of

Serum Cholesterol> 500 mg/dl

n=12

Serum Cholesterol< 5OO mg/dl

n=8

Serum protein (g/dl)

Creat inine (mg/dl)

Blood Urea (mg/dl l

Urinary protein (g/L)

lmmunoglobul ins (mg/dl) :lgGlgAlgM

5.O8 rO.241.OB +0.19

45.5 r 16.7*7.6 r 1 .O*

54.06 r 5.58*168.4 r .11.25230.2 + 21.3

5.1r0.321.03 r0.2532.6t7.63.62 +0.9

1 15.1 + 13.18179.3 * 19.9169.5 + 23.6

Values are rnean t SEMLevels of s igni f icance compared to controls (Student 's t- test l : *P(0.05

References1. Giangiacomo J, Cleavy TG, Cole BR, Hoffsten, P. o

and Robson, A.M. (1995) N. Engl . J. Med., 293 : - ' Doumas, B.T., Watson, W.A. and Biggs, H.G.(19971 Clin. Chim. Acta, 258 t 21 .

Merle, A.E. (19981 ln Tietz Textbook of ClinicalChemistry. (Eds. Burtis, C.A. and Ashwood, E,R.lWB,Saunders Co., Philadelphia and Singapore, p.75.

Maucini , G., Carbonara, A. O. and Heremaus, J,F.f l9651 lmmunochemistry, 2 : 235.

Ogi, M, Yokoyana, H., Tomosugi, N., Hisada, Y.,Ohta. S. , Takaeda, M. et a l . (1994). Am. J 'Kidney Dis,, 24 :, 427.

Waldnerr , R,, Gubles, M.C. and Levy, M. (1983)Kidney lnt., 23 t 368,

Fechal ly, J. , Beatt ie. T.J. and Brendley, P.E.C.(1984) N. Eng. J. Med.,31O:415.

Zel leruelo, G., Hsia, S. L. , Freundl ich, M.,Gorman, H.M. and Strauss, J. (19841. J. Pediatr.,104:61.

L

Shalhoub, R.J. (1 9741 Lancet 2 : 556.

Andal , A. , Chel lani , H., Anand, N.K. and Chandra,M. (199O1 lndian Pediatr., 27 : 1O45,

Chen, C.H. (1987) lnt. J. Pediatr. Nephro.,8 : 75.

Marks, V. (19881 ln Pratical Clinical Biochemistry,(Ed, Varley, H.) 4th ed. Reprint CBS Publishers andDistributor, India, p. 80.

Newman, D.J. and Price, C.P. Renal function andnitrogen metabolites. ln Tietz Textbook of ClinicalChemistry, (Eds. Burtis, C.A. and Ashwood, E.R')3rd ed. WB Saunders Company, Philadelphia andSingapore p. 12O4.

Kingsfey, G.R. (19421J. Lab. Clin. Med., 27 :84O.

Doumas, B.T. and Peters, T. Jr. (1997l. Clin.Chim. Acta,258 : 3.

10.2.

3.

4.

5.

7.

6.

11.

12.

13.

14.

8.

15,

brd. J. Med. Biochem., 2OO1, 5(1&21

Study on Free Radicals in Type ll Diabetes Mellitus

Chhanda Basu Mull ick and Sandip Bandopadhyay*Department of Biochemistry, Medical College and

*University College of Medicine, Kolkata

Abstract72 follow-up cases of NIDDM including 43 well-controlled and 29 i l l-conirolled diabetics wereassayed for l ipid peroxide level in the form of Thio Barbituric Acid Reacting Substances (TBARSI asa measure of oxidative stress against 25 age-matched healthy controls. Mean plasma TBARS levetwas significantly greater in well-controlled diabetics than healthy controls ( p < O.O5l and this wasmore accentuated in poorly controlled diabetics (p< O.OO1l. Mean plasma TBARS levet in 3gpatients with long term vascular complications las obtained from the 72.type i l diabetic cases) washigher than age-matched healthy controls as well as diabetics without complications. The studysuggests that plasma TBARS level might be used as an adjunct to glycemic status in the evaluationof NIDDM patients and progression of its complications.

IntroductionFree radicals arc reactive biochemicalintermediates that have been increasinglyimplicated in the pathogenesis of Diabetesmellitus. Being extremely reactive, they aremostly elusive to measurement by direct assay inthe relevant ctinical context.r'2 Consequently,semiquantitative assays are mostly util ised forindirect measurement of end products of freeradical damage to broad classes of biomolecules,namely, l ip ids, proteins and nucleic acids2.

ln this study oxidative stress due to free radicals,causing membrane lipid peroxidation is measuredby assaying thiobarbituric acid reactingsubstances including malondialdehyde (TBARSI inthe plasma of 72 NIDDM patients with or withoutcompl icat ions.

Patients and MethodsIn this cross-sect ional study, fol low-up cases of72 NIDDM (with or without compl icat ions)anending the Medicine and Endocrinology OPD ofSSKM Hospital, Calcutta were clinically reviewed

and their lipid peroxidation products in the formof TBARS were semiquantitatively assayed at theDept. of Biochemistry, University College ofMedicine, Calcutta and compared with 25age-matched controls during the period August '1995 to July 1996.Seventy two patients, over 40 years of age,suffer ing from NIDDM for more than 2 vrs. ,having no other associated diseases and receivingno other treatment other than diabetic diet withor without oral hypoglycaemic drugs or insul inwere included in the study. NIDDM wasdiagnosed on the basis of persistent

hyperglycaemia along with classical symptoms ofpolyur ia, polyphagia and polydipsia.

Cl inical prof i le of the pat ients including age, sex,family history, medicat ion, history of smoking,relevant signs and symptoms of diabetic macro-and microvascular compl icat ions were noted.NIDDM pat ients were f i rst div ided aswel l-control led and i l l -control led on the basis ofhyperglycaemia and then stat i fed according toage into two, > 55 yrs and < 55 yrs. Amongthe 72 pat ients 38 pat ients (13 wel l -control led

Ind. J. Med. Biochem., 2OO1, 5(1&21

and 25 i l l -control led) showed vascularcompl icat ions, data of which were calculatedand seperately. Details of classificeationspresented are shown in Table 1. Twenty fiveage-matched healthy individuals working indifferent departments of University College ofMedicine were taken as controls.Plasma glucose estimation was done by glucoseoxidase peroxidase method (Span Diagnostics)and measured spectrophotometrically at 505 nm(Chemite 21O0 - Scanning UV - VIS). HbA," wasestimated by affinity chromatography usingvenoLis EDTA blood wherefrom RBC washemolysed (by Triton X ) for estimation. PlasmaTBA,RS including malondiadehyde was estimatedfollowing a modified method3'a by usingthiobarbituric acid reagent at 532nm and valueswere expressed as pmol/L malondialdehydeequivalent. Standard used was 1 ,1 ,3,3tetraethoxypropane (TEP malondialdehyde, SigmaChemicals, USA).

ResultsFast ing plasma glucose, post prandial plasmaglucose and glycosylated haemoglobin of theNIDDM pat ients.are shown in Table 1. The serumlevels of TBARS including MDA of the NIDDMpatients and control are presented in Table 2. Theresults revealed that TBARS was higher in all thepatients as compared to that of correspondingage-matched controls. However it was evenhigher in il l-controlied. patients with a highest

value in the age group of above 55 yrs. Thepatients with vascular complications also showed

significantly higher value while the elevation in

the TBARS level was not significant in

well-controlled patients as compared to that of

controls.

DiscussionDiabetes is a comolex metabol ic disease andmany factors are implicated in its long-term

complications. Though normalisation of glycaemic

8

status is the primary therapeutic strategy in thecontrol of diabetes and its complications (DCCTtrial), y€t, the occurence of macrovasculardisorders including peripheral vascular disease,atherosclerosis, ischaemic heart disease andischaemic cerebrovascular disease andmicrovascular disorders like neuropathy,nephropathy and retinopathy should beconsidereds. Oxidative stress due to free radicalgeneration has been increasingly evidenced tounderly the core of pathogenesis of diabeticstate. lt is often responsible for the diabeticcomplications irrespective of glycaemic statusdue to variability in ihdividual resistance to thisstresss. The effect of free radical injury can bemeasured in the form of various products of lipidpetoxidation (alkanes, alkenes, dicarbonyl,saturated and unsaturated aldehydes i.e.malondialdehyde in the form of TBARS,4-OH-nonenal, ethane and pentanes) or productsof protein damage (carbonyl group assayl or DNAdamage6. TBARS is used as a general indicator ofoxidative stress resulting in lipid peroxidation.The role of oxidative Etress in diabetes and its'complications is supported by many hypotheseswith experimental back-up, like thealdose-reductase hypothesis, proteinglycosylation and monosaccharide autooxidationhypothesis etc.7. Earliers'ro studies reported thatwhole plasma lipid peroxide levels in the form ofTBARS in diabet ics was higher than in controlsand is highest in those with angiopathy, and thisis appl icable both in IDDM and N|DDMrr.Sato et alr0 reported a statistically significantincrease in TBA-reactivity by fluorimetric methodin diabet ics than in controis in a study of 1 10diabet ics against 331 controls of average 40-50years age-group. 'The TBARS levels in those with

angiopathy was signi f icant ly much higher than in

those without angiopathy, whose levels werealmost comparable to normals. They also noted aplasma TBARS level of 6.04 1.11 n mol/ml inpoorly - control led diabet ics as against

Ind. J. Med. Biochem., 2001, 5(1&21

4.04t0.37 n mol/ml in wel l -control led pat ients.Gallon and co-workersr2 also demonstrated anincrease in lipid peroxidation (by measuringTBARS) in diabetic patients.Although measurement of MDA in the form ofTBARS has been a well established indicator oflipid peroxidation, measurement of 4-OH-nonenalis more specific for lipid peroxidation but hard io

9

use in cl in ical t r ia l t3. Recent ly, ant ibodies havebeen used to detect MDA modified proteins and4-hydroxynonenal modified proteins with help ofRIA and ELISA which may serve better for in-vivodetection of lipid peroxidationto. Whatever be themethod, the measurement of TBARS along withthe glycaemic control could be used profitably asan early indicator of vascular compl icat ions indiabetics.

Table 1. Fasting plasma glucose (FpG), post-parandial plasma glucose (pppcl and glycosylatedhaemoglobin (HbA," l in NIDDM pat ients.

NIDDM patients n Mean FPG (mg/dll Mean PPPG (mg/dl) Mean HbA,. (7o)

Well.controlled <55 yrsWell-controlled >55 yrs

Itt-controlled <55 yrsI l l -control led )55 yrs

25181019

125138162212

178190180320

5.27.210.512.6

The range of FPG and PPPG in contols were 8O-100 mg/dl and 12O-140 mg/dl respectively and HbA," 17o/o.

Tabte 2. TBARS level (pmol/Ll in NIDDM patients and controls.

TBARS LevelNIDDM patient ( 55 yrs. ) 55 yrs.

3.14rO.545.79 +O.7*

4.97 t 1.33*1.99rO.80

Values are expressed as mean tSE; Lovels of s igni f icance as compared to controls: f p<O.OO1

Well-controlledIll-controlled

With complicationsControls

43293825

2.80*0.484.96 r 1.O1 *

4.32i ,1.49!1 .56 r 0.45

References1. Holley, A.E. and Cheeseman, K.H. (19931 Ar.

Med. Bull.,49 : 494.

2. Slater, T.F. Methods,Enzymol., 105 : 283.

3. Packer Lesler (19841 ln Methods in Enzymology,

Academic Press, New York, Vol. 105, p. 286.4. Das, U.N., Vijay Kumar, K. and Krishna Mohan, l.

(1994) J. Nutr. Med., 4 : 149.

5. Wofff, S.P. 119931 Br, Med. Butt., 49 : 642.6. Pryer, W.A. ond Godber, S.S. (19911 Free Radic.

Biol. Med., 1O : 177,

7. Cheesemtr, K.H. and Slater, T.F. t1 9931 Br. Med.

8ull., 49 : 481.

8. Nishigaki, 1., Hogihara, M., Tsunekawa, H, et al.(19811. Biochem. Med., 25 : 373.

9. Uzel , N., Sivas, A., Uysal , M. and Oz, H. (1987).Horm. Metals 8es., 19 : 89.

10. Sato, Y. Hotta, N., Sakamoto, N., Matsuoko, S.,' Ohishi , N. and Yagi , K. (19791. Biochem. Med.,21

:104.1 1. Kaji, H., Kurasak, M. l. and Tu, K. 119851 Klin.

Wochen.Schr., 63 I 765.12. Gal lon, G., Ruel land, A., Legras, B. et a l . (19931

Clin. Chem. Acta, 214 : 227.13. Estrabauer, H., Schaver. R.J. and Zol lner, H.

{19911 Free Rad. Biol. Med., 1 1 : 81.14, Amara, A., Constans, J. , Changier, C., Sebban,

An, et al (1996). Clin. Exp. lmmunol., 101 : 233.

Ind. J. Med. Biochem., 2OOl, 5(1&21

Hepatoprotective and Hypoglycaemic Effects of Phyttanthus niruri

R. Sanjenbam, H.B. James, S. Akham and M.A. Singh*Regional Inst i tute of Medical Sciences, lmphal, Manipur

*Department of Pharmacology and Department of Biochemistry

AbstractThe hepatoprotective and hypoglycemic activit ies of aqueous extract of the dried leaves ofPhyllanthus niruri was investigated. Hepatotoxicity was induced in rats by Carbon tetrachloride(CCl.) and hepatoprotective activity was measured by determining serum alanine trangaminaee(ALTI ans aspartate transaminase IASTI and compared with silymarin as standard. Hypoglycemiceffect was evaluated on alloxan induced diabetic rats by the estimation of blood glucose byglucose oxidase method and compared with glibenclamide as standard. Aqueous extract of P.nirurishowed significant hepatoprotective and hypoglycemic activit ies in rats. Hepatoprotective property

may be due to the presence of l ignan compounds, which inhibit peroxide formation and scavengefree radicals. The hypoglycemic property on alloxan induced diabetic rats may be due to thepresence of f lavonoids which has hypoglycemic activity.

10

IntroductionPhyllanthus niruri is a common herb growing

abundant ly in the hi l ls and val leys of Manipur andother parts of the world. The plant has beenwidely used by traditional medicinal practitionersfor the treatment of stomach troublesr, colickypain, fevers2, dysenterys and jaundice 6nddiabetes mellitus2'a Systemic research on anypossible effect of P. niruri. on CClo - inducedhepat ic damage and al loxan - induced diabetesmel l i tus2'a seems to be scarce, and in view of i tsreputed use in liver ailments the aqueous extractof P. niruri was taken up for investigation ofhepatoprotective and hypoglycaemic properties inexperimental animals.

Mater ials and MethodsFresh leaves of the plant were col lected during

the month of June and July, dr ied under shadeand powdered. Forty grams of the powdered

leaves were boi led with 2 l i t res of dist i l led waterfor 20 minutes, cooled and f i l tered through clear

musl in cloth. The f i l t rate was dr ied and used forthe test6.The fol lowing chemicals and drugs from variousmanufacturers were used : 306 Tween 80 (LOBA

CHEMIEI - 0.75 ml/kg per oral as a vehicle,Carbon Tetrachlor ide (CClo) - 0.75 ml/kg per oral ,Syl imarin (MICROLAB) - 50 mg/kg per oral , ALTand AST est imation ki t (RANBAXY

LABORATORIESIand test drug - 2OO mg/kg and5OO mg/kg per oral , Al loxan monohydrate - 150mg/kg i .p and Gl ibenclamide (HOECHSTI - 0.5g/kg per oral .Adult albino rats (Wister Strain) of either sex,

weighing between 200-250 g were selected for

the experimental study. Aninials were dividedinto 7 groups, each consist ing of 5 rats and keptunder standard laboratory condit ions. They had

free access to commercial pel let diet and water.

The protocol for the assessment of

hepatoprotect ive act iv i ty against CCl4 - induced

hepatotoxici ty is given in Table 1. The rats were

divided into control , toxicant, standard and testgroup, The control and toxicant groups received

lnd. J. Med. Biochem., 2001, 5(1&21

the vehicle whi le the test and standard groupsreceived the f i rst dose of respect ive suspension30 minutes before intoxicat ion with a single oraldose of CClo. The standard and test groupsreceived two doses after 12 hours and 24 hours.Blood was col lected after 36 hours f rom theorbi tal s inus of albino rat and enzyme est imationswere done. Three groups of rats were keptf ast ing for 24 hrs and al loxan monohydrateprepared as aqueous solut ion in normal sal inewas given intraperi toneal ly at a dose of 150mg/kg body weight. Diabet ic rats were selectedby est imation of blood glucose by glucose

oxidase methods. Rats with blood glucose in therange of 25O-4OO mg were taken lor theexperiment. Fast ing blood glucose levels of al l thediabet ic rats were est imated in the morningfol lowed by administrat ion of the test drug(aqueous extract of P. niruril, standard drug(Gl ibenclamide) and aqueous 3% Tween 80 toeach of the three groups respect ively. After 2 hrs

of administrat ion of the drugs, blood was drawnfrom the orbi tal s inus and blood glucose wasest imated again.The data were stat ist ical ly analysed by employingunpaired students " t" test. P values less thanO.O5 were considered to be signi f icant and valuesless than O.O1 were considered highly signi f icant.

ResultsSerum transminase act iv i t ies of animals used for

hepatoprotect ive eff icacy are shown in Table 2.

Rats treated with CClo (Toxic Control) showed

signif icant increase in serum ALT and AST levels

compared to the control . Oral administrat ion of

the water extract of P. niruri (2OO mg/kg)

showed a s igni f icant reduct ion (P<O.01) in thelevels of ALT and AST. Si lymarin produced more

signif icant reduct ion (P< O.O01l in ALT andAST. ln the al loxan - induced diabet ic rats (blood

glucose: 250-400 mg/dl) P. niruri. water extract(500 mg/kg) reduced the fast ing blood glucose

level f rom 3O2 mg/dl to 218.2 mg/dl and the

11

reduct ion in gl ibenclamide treated group wasfrom 290.4 mg/dl to 73.2 mgldl at ter 2 hours.The reduct ion in blood glucose in both the groups

were signi f icant. The serum glucose level in

diabet ic rats before and af ter administrat ion ofthe test drug are given in Table 2.

DiscussionHepat ic necrosis induced by CClo leads to prompt

increase in serum enzyme markers al lowing

detect ion of ear ly damage without the need to

sacr i f ice the animals. Est imat ion of ALT and AST

ate the most sensi t ive tests employed in

diagnosis of hepat ic disease. Si lymarin, a

flavolignan compound of Silybum marianum, is a

mixture of s i lybin, s i ld ianin and si lycr ist in. The

hepatoprotect ive propert ies of s i lymarin have

been related to inhibi t ion of l ip id peroxide

format ion or scavenging of f ree radicals. In the

present study, P. niruri. water extract

s igni f icant ly reduced the higher levels of ALT and

AST induced by CCl4 as compared to the

controls. The hepatoprotective property of P.

niruri could be due to the presence of l ignan

compound which can inhibi t peroxide format ion

and scavenge free radicals as evidented in ear l ier

studiesT's ' r r .

The ef f ect of a s ingle administrat ion of the

aqueous extract of P. niruri at a dose of 500

mg/kg produced signi f icant reduct ion of b lood

gfucose (P<O.O2l in diabet ic rats at 2 hrs,

al though i t was found less ef fect ive than that of

gl ibenclamide. The hypoglycaemic ef fect of P.

niruri may be due to the presence of

phytochemicals such as f lavonoids which have

hypoglycaemic act iv i ty.

Thus, the aqueous extract of P. niruri induced

interest for the studies to isolate ant ihepatotoxic

and hypoglycaemic pr inciples which wi l l be

benef ic ia l for the preparat ion of low-cost herbal

drugs.

Ind. J. Med. Biochem., 2OO1, 5(1&2)

Table 1. Protocol for hepatoprotect ive study.

12

Group O hrs. 1/2 hrs. 12 hrs. 24 hrs. 36 hrs.

ControlToxicantStandardTest

VehicleVehicle

Si lymarinTest drug

Normal salinecc14

VehicleVehicle

SilymarinTest drug

VehicleVehicle

Si lymarinTest drug

cc14ccl4

Enzyme estimationEnzyme estimationEnzyme estimationEnzyme estimation

Tabfe 2. Hepatoprotective and hypoglycaemic activities of P. niruri in albino rats.

Groups

ControlToxic ControlStandardTest

AST(U/L}

305241241444426271b72748'

ALT(U/LI

914311 3575641 1 8gb

67852"

Levels of significance as compared to corresponding controls:

'P<0.O2; bP<O.O1 ; "P<O.OO1

References1. Kirtikar, K.R. and Basu, B.D.|19871. ln lndian

Medicinal Plants., Vol- lll. E. Blatter, Caius J.F.

and Mhaskar K.S. lEdn.l lnternational Book

Distributors, Dehradun, p.225.

2. Sinha, S.C. (1996) Medicinal Plants of Manipur,

Sinha, SC (Edl Association for Science and

Society, Manipur, p.1 40.

3. Kurian. J.C.(l9951 Phyllanthus, Plants that heal,

Oriental Watchman Publishing House, Pune,

p.247.

Mathur, S. Devi, 1., Kumar, D., Sahay, B.K. and

Begum. F.(1989). Indian J. Pharmacol.,21z5O.

Agarwal, S.L., Deshmankar, 8.S., Verma, S.C.L.

and Saxena, S.P.(19601 lndian J. Med. Bes,.,48 :

457.

Groups Blood glucose (mg/dll

Before drug After drug

Diabetic control 341 .4 t21 .43 329:0 + 25.58Test 3O2 .8 t 21 .28 2 1 8.O r 24.64'

Standard 280.4* 21.19 73.2*,1 1.37b

6. Kurna, S.R and Mishra, S.H. 119961. lndian

Drugs,33 :456.

Rao, K.S. and Mishra, S.H. {19971. lndian J.

Pharmacol., 28 : 1 10.

Brahans, D. and Trinder, P. (19721. Analyst., 97 z

142.

Bindole, A., Cowallini, L. and Sil ipandi, N.(19971.

Biochem. Pharmacol., 26 :24O5.

Shyamasunder, K.U., Singh, 8., Thakur, R.S,

Hussain, A.,Kiso, Y. and Hikino, H.(19851 J.

Ethnopharmacol., 14 241 .

Mondal, B., Bhattacharjee B. and Maity,

C.Rl1999l lnd. Med. Biochem.,3 :17.

7.

8.

9.

10.

11.

4.

5.

nd. J. Med. Biochem., 2O01, 5{1&2}

Diagnosis of Falciparum Malaria by lmmunochromatographic Test

Lt Col Parduman Singh, Lt Col D Mitra and Maj (Mrs) Sonia Badwal155 Base Hospital, C/O 99 APO

AbstractLimitations and advantages of direct microscopy and newer methods for detection ol plasmodium

falciparum malaria like Fluorescence techniques, PCR techniques and antigen captive assays havebeen discussed. 154 patients with clinical features suggestive of malaria were studied to evaluatethe efficacy of immunochromatographic test (lCT), which detects histidine rich protein-2 antigensecreted by Plasmodium falciparum (pf HRP-2), as against direct microscopy. There were 30 casesol P, falciparum malaria, 1 case ol P vivax malaria, 1 case of mixed infection with P. falciparumand P. vivax and 122 cases of non-malarial fever. Direct microscopy could detect 8(26.60/ol p.

falciparum cases but failed to detect 22 cases (73.4olol whereas ICT could detect 29 196.7o/ol p.

falciparum cases out of 3O but failed to detect 1 (3.3%l P. falciparum case P. y/yax cases andnon-malarial fever cases were negative for lCT. The sensitivity and specificity of ICT is 96.7% andl OOo/o respectively. lt is concluded that ICT test is a good adjunct to blood smear studies in feverwith neurological and multiorgan dysfunction.

13

Introduct ionRecent data suggest malar ia being responsible forabout 2 mi l l ion deaths per year in the world.Every year 3OO-5OO million persons contract thisdisease with unmeasured impact on economy,human health and longevityr. Accurate diagnosisis the corner stone for proper management ofmalar ia and to prevent compl icat ions. Malar iadiagnosed on the basis of c l in ical symptomsalone is at best 50% accurate2. Therefore, therole of laboratory is to give precise and rapiddiagnosis. Direct detect ion of malar ial parasi te bymicroscopy remains the gold standard fordiagnosis of malar ia. This technique is simple,reproducible and cost ef fect ive. Thin and thickperipheral smears stained with Giemsa areexamined f or malar ial parasi te. Thick smearsprovide the sensit iv i ty and thin smears give thespecif ic i ty to the technique. Specif ic i ty of thinsinear is much better than the thick smear forspecies ident i f icat ion and evaluat ion of the

intensity of parasi temia3. However diagnosisusing this method can be unsat isfactory since i ttakes about one hour of preparat ion t ime andsample may not always be collected timely duringthe febr i le phase. l t is labour intensive andinterpretation of the results requires considerableexpertise, especially at low levels of parasitemiaa.

f n addition, in patients with P. falciparum malaria,there may be sequestration of the parasites,

which may not always be present in peripheral

blood. Whi le screening large number of smears,chances of human error are highs. Considering

these l imitat ions, al ternate techniques for thediagnosis of malar ia have been developed. These

newer techniques consist of f luorescenttechniques, PCR techniques and Ant igen Capt iveTests.Three types of f luorescent techniques are invogue for the diagnosis of malar ia. Ouant i tat iveBuffy Coat Assay (OBC Methodl is avai lable as a

commercial k i t . Two other techniques are

Ind. J. Med. Biochem., 2O01, 5(1&2)

Kawamoto Acridine Orange Process6 andBenzothio Carboxy Purine (BCP) procedureT.

These three techniques are rapid and relat ivelyeasy to perform and demonstrate sensit iv i ty andspecif ic i ty equivalent to that achievable byexaminat ion of stained thick smear. OBC andKawamoto methods use Acridine Orange (AO) asf luorochrome to stain the nucleic acid of themalar ial parasi te. BCP is also a f luorochrome,which stains nuclei acid. lmportant l imitat ion ofthese methods is their inabi l i ty to di f ferent iatePlasmodium species. lnspite of their l imitat ionsl ike requirement of special staining and expensiveequipment, the f luorescent microscopy for rapiddetect ion of malar ial parasi te in blood is a viablealternat ive to examinat ion of Giemsa stainedsmears.Another approach to the laboratory diagnosis ofmalar ia is based on the detect ion of nucleic acidsequence specif ic to Plasmodium species usingPCR technique. This technique can permit speciesspecif ic diagnosis of Plasmodium infect ion andcan also detect mixed infections. The majoradvantage of using PCR based technique is in i tsabi l i ty to detect infect ion in pat ients withparasi temia as low as 5 parasi tes/microl i t re with100% specif ic i tye. However the technique is

expensive and labour intensive, requires

extensive technical expertise, involves multiplesteps and cannot be used to dist inguish betweenviable and non-viable organisms. PCR inhibi torsnatural ly present in the blood may result in

signi f icant number of false negat ive results. Falseposit ive results due to carrying over

contaminat ion have also been recordede.Antigen Captive Tests are capable of detecting

fewer parasi tes at a faster speed. One of these

tests can dist inguish viable f rom non-viableparasi tes. These tests are a promising tool for

monitor ing ant i-malar ial therapy. Hist idine Rich

Protein-2 (HRP-2) and Parasi te Lactate

Dehydrogenase (pLDH) are two parasi te ant igens

current ly used in these new, rapid diagnost ic

14

tests. HRP-2 is only produced by P. falciparum'oand pLDH ant igen is produced by al l four speciesof Plasmodium which infect man. Both theseant igens are produced by asexual stages of theparasi te whereas pLDH is also secreted bygametocytesrr.

Ant igen Capt ive Tests are rapid and simple toperform and have detect ion l imits comparable 1othose of high qual i ty microscopy i .e. 1OO-200parasi tes/microl i t rer2. OBC assay and ant igencapt ive tests can be recommended as f i rst l inediagnost ic choice for malar ia whereas ut i l i ty ofspecies specif ic PCR is in diagnosis of falc iparummalar ia. They are part icular ly useful for.studies ofstrain di f ferences, mutat ions and genes involvedin drug resistance rather than for rout inediagnosis. Out of these tests, the most promising

diagnost ic tests are serological commercial lyavai lable dipst ick tests l ike ParaSight F,ParaCheck and Rapid-MP which are based onHRP-2 ant igen and OptiMAL test based ondetect ion of pLDH ant igen.We have undertaken a study at Mi l i tary Hospital ,Tezpur (Assam) to compare the diagnost iceff icacy of direct microscopy withimmunochromatographic test which detectsPlasmodium falciparum HRP-2 (pfHRP-2) ant igen.

Mater ials and MethodsThis prospect ive study was undertaken at Mi l i taryHospital , Tezpur (Assam) to compare the

accuracy of the two readi ly avai lable methods for

diagnosis of P.falciparum infection, directmicroscopy and detect ion of pf HRP-2 by an

immunochromatographic test ( lCTl . A total of

154 pat ients were studied who had cl inical

features suggest ive of malar ia with or without

splenomegaly, central nervous system

involvement or systemic compl icat ions. The thick

and thin Giemsa stained blood smears were

screened two t imes fol lowed by an lCT. Fresh

blood f rom the pat ients was col lected in

EDTA/heparin/oxalate and the test carr ied out as

. rc. J. Med, Biochem.,2001,5{1&2}

Jer the recommendations of the manufacturers.T;re ki ts named ParaCheck from Orchid3iomedical Systems were used. In this test twoant ibodies specif ic for pfHRP-2 ant igen are used.One of the ant ibodies is attached to a vis iblecoi loidal gold impregnated into sample pad, whi lelhe second ant ibody is immobi l ised as a l ineacross the test str ip. 5 microl i t re of whole bloodis added to the sample pad where lysis occursand pfHRA-2 ant igen, i f present, binds to thecol loidal gold lablel led ant ibody. When a givenreagent is added to the sample pad, blood andlabel led ant ibody migrate towards test str ipcrossing the second ant ibody l ine. Appearance ofa pink l ine indicates binding of pfHRP-2 to secondantibody and thus presence of P. falciparumant igen.

ResultsA total of 154 pat ients, who presented withfever and other signs and symptoms suggest iveof malar ia, were studied. The diagnosis of malar iawas conf irmed in 32 pat ients (Table- l) . Out of3O cases diagnosed as P. falciparum malaria 7were positive both by direct microscopy and lCT.8 (26.60/0l cases were P. falciparum positive bydirect microscopy. ICT was posit ive in

29(96.7%l P. vivax was present in one case, andone case had mixed infection of P. vivax and P.falciparum. In none of the 122 non-malarialfevers and P. vivax case ICT was positive,

account ing for lOOo/o specif ic i ty. But i t fa i led to

detect 1 smear posit ive case account ing f or

96.7o/o sensitivity. The positive predictive value

of the test was 1OO% and the negat ive predict ive

value was 96.7%.

DiscussionIn this study the specif ic i ty and sensit iv i ty of ICT

was found to be 100% and 96.7o/o respect ively.

Dif f erent studies in India have specif ic i ty and

sensi t iv i ty ranging between 87 .5 - 1OO%(Table-2). Our f indings are closer to the study,

15

conducted by Mishra et alr3. lCT, in our study,had a posit ive predict ive value of lOOo/o andnegat ive predict ive value of 96,7o/o as comparedto 98.7% and 1OO% respect ively reported byKumar et alra and 1OO% and 87.5o/o respect ivelyreported by Gupta et al 's. The study by Kar et alhas reported both sensitivity and specificity as100%r0.Though smear posit iv i ty is considered as the goldstandard, i t is wel l documented that in somecases the parasi te is local ised only in internalorgans. In 7 of our pat ients, there was enoughcfinicaf evidence of P. falciparum malaria, such asfever with chi l ls and r igor, hepato-splenomegaly,central nervous system and multiorgandysfunct ion. But their per ipheral blood smearswere repeatedly negative f or P. falciparumthough ICT was posit ive. Al l of them respondedwel l to ant imalar ial t reatment.One case was smear positive tor P. vivax but ICTwas negative and one case was smear positive

both for P. vivax and P. falciparum and ICT wasalso posit ive. ICT was helpful in the diagnosis ofP. falciparum infection in cases of f ever withshock, seizures or abnormal behavior where theparasi temia may be ei ther low or absent as theparasi tes may be sequestrated in internal organs.ln such cases the ant igen can be detected by ICTand treatment inst i tuted successf ul ly. Thus i tmay be concluded that ICT woi.rld be a good

adjunct to blood smear studies part icular ly for thedetect ion of falc iparum malar ia.

Ind. J. Med. Biochem., 2001, 5(1&2) 16

Table 1. omparison of blood smear with ICT for the detection of P. fatciparum and P. vivax malaria

Case Type Number Blood Smear ICT Test

Posit ive Negat ive Posi t ive Negat ive

P. falciparum 30 8 l26.6vol 22 '73.4o/ol 29 (96.7%1 1 (3.3%)

P. vivax 1

122Non malar ia l 122 Ocases

1r 1

122 o

Total 154 9 145 30 124* Mixed infection with P. vivax and P. falciparum.

Table 2. Comparison of sensi t iv i ty and speci f ic i ty among var ious studies fo l lowing ICT

Studies Sensi t iv i ty (%) Speci f ic i ty (%)

Mishra et a l t3

Kumar et a l t4

Gupta et a l ts

Kar et all6

Present Study

97100

87.510096.7

10098.7100100100

References

1. But ler D. (1997) Nature 386:609. 10. Howard, R.J. , Uni , S. , Aibawa, M., Aley, S. , Leech.

2. WHO (19961 Bul let in of WHO 74 : 47. J.H., Wel lems, T.E., Rener, J. , and Taylor, D.W.

3. Warhurst , D.C. and Wil l iams. J.E. (19961 J. Cl in. (1986). J. Cel l . Biol , 103:1269.

Pathol . ,49:533. 11. Oduola, A.M.J, , Omitonoju, G.D., Sowunmi, A, ,

4. Nlabandin, R.M., Sammons, D.W., Manl ly, M.8. , Xie, Mabler M.T., Falade, C.O,, Kyle, D.E., Fehintola, F.A.,

L. , Ster l ing, C.R., Egen, N.8. , and Gl ingras, B.A. (19951. Ogundahunsi , A.O.T., Schuster, B.E. and Mi l tous, W.K.

Am. J. Clin. Pathol. 103 : 57. (19891 Exp. Parasitol.,87 z 283.

5. Baird, J.K., Purnomo and Jones, T.R. (19921 Trans. 12. Palmer, C.J. , L indo, J.F. , .Klabala, W., Quesada, J.

19.Soc. Trop.Med.Hyg.86:3-5. andAger,A.L.(19981J. Cl in.Microbiol . ,36:2O3

6. Kawamoto F. (19911 Lancet 1 : 2OO. 13. Mishra, B. , Samantaray; J.C. and Nirdha, B.R.

7. Mabler, M.T., Ries, J, Harton, R.J. and Hinr ichs, D.J. (19991 lnd.J. Med.8es., 109: 16.

{1991} Am.J. Trop. Med. Hyg.,44:11. 14. Kumar, A. , Sharma, V.P., Thavaselvam, D' and

8. Barker, R.H. (Jr . ) (1990) Exp. Parasi to l . . , 170:.494. Sumodan, P.K. (19961 lnd. J. Malar io l . ,33 : 166.

9. Kawamoto, F, Miyabe, H, Kanebo, O, Kimura, M., 15. Gupta, M.K., Mishra, R.N., Chawla, N., Mani, H"

Nguyen, O., L iu, T.D., Zhou, M., Le, DD., Kawai, S. , Chowdhry, C'N., Singh, S'P. and Gupta, S. (2OO1l

lsomura, S. and Wataya, Y. (1996) J. Cl in. Microbiol . , MJAFI 57:188.

34 : 2287. 16. Kar, 1. , Eapen, A., Adah, T' and Sharma, V.P.(1998)

lnd. J. Malariol., 35 : 160.

,nd. J. Med. Biochem.,2001,5(1&21

The Effect of Thyroid Diseases on Humoral lmmunity

Vikram Kesar, B.D. Banerjee, A.K. Chakravarty and R. Avasthi ' ,Departments of Biochemistry and Medicine'

Universi ty Col lege of Medical Sciences and G.T.B. Hospital ' , Delhi - 11OOgs

AbstractSerum immunoglobulins, lgG and lgM were measured in 2O patients suffering from hypothyroidism,20 patients of hyperthyroidism and 2O healthy controls. Serum lgG concentration was significantlyincreased in hyperthyroid patients although it was within the normal physiological range.Hypothyroid patients had significantly lower serum lgM concentration but it was stil l within thebiological limit. Serum lgG level in hypothyroid patients was found to be significantly reducedbeyond the reference range. The results indicate a slight depression ol humoral immunity inhypothyroidism which might be responsible for the increased incidence of infection in thesepatients.

17

IntroductionThyrotropin (TSH) was one of the first hormonesrecognised for i ts role in immune regulat ion andis known to enhance the in vi t ro ant ibodyresponse at physiological concentrat ion. Al thoughTSH is a pi tui tary hormone whose product ion isst imulated by thyrotropin releasing hormone(TRHI and suppressed by thyroid hormones (T,

and To ), i t is also known to be produced by Tcel ls in response to the same st imul i r .The inf luence of thyroid dysf unct ion on theimmune system of humans has not been clear ly

def ined. Animal studies indicated decreasedlymphocyte funct ion, reduced thymus weight anda decrease in the number of total WBC's inhypothyroid chickens. Thyroxine treatment

usual ly enhanced thymus weight and causedlymphocytosis in :experimental animals2.

Stimulatory effect of thyroid hormones on cell

mediated immunity (CMl) has also been

demonstrated in rats3.A study of thymul in levels in hypo-and

hyperthyroid pat ients also provides evidenceregarding the regulat ion of immune funct ion by

thyroid hormones. The thymus produces a

hormone-l ike substance cal led thymul in that

induces prol i ferat ion and di f ferent iat ion of T cel ls.Hyperthyroid pat ients had higher thymul in levelwhi le hypothyroid pat ients had lower thymul inlevels in comparison to normal subjectsa.Hypothyroidism results in impaired immuneresponse to most types of infections withpulmonary and genito-ur inary tract infect ionsbeing most common in these pat ients. Severelydecreased lymphocyte funct ion has been reportedin a hypothyroid patient with bacteraemias.Human lymphocytes carry nuclear receptors forthyroid hormones which may exert their act ionvia these nuclear receptorsG. The present studywas undertaken to study the serumimmunoglobul ins ( lgM and lgGl in hypo-andhyperthyroid pat ients.

Mater ials and MethodsThe study was conducted with 60 subjects,

consist ing of 20 hypothyroid pat ients, 20

hyperthyroid pat ients and 20 normal healthy

controls of the same age group. A detai ledhistory f ol lowed by a general physical and

systemic examinat ion was performed on al l the

subjects in the medical /endocinology O.P.D. of

G.T.B. Hospital , Delhi . The hypothyroid cases

Ind. J. Med. Biochem., 2O01, 5(1&21

were selected based on the f inding of subnormalserum T4, T3 and elevated serum TSH. Simi lar lythe hyperthyroid cases were selected on thebasis of increased serum To, T. and decreasedserum TSH levels. Pat ients having any act ive orchronic infect ion or inf lammation which couldaffect the serum immunoglobul in level wereexcluded from the study. Heparinised bloodsamples were col lected for the est imation ofserum lgG and lgM using tr i -part igenimmunodif fusion plates (Hoechst India Ltd. l . Theprinciple of determinat ion is based on single radialimmunodif fusion of immunoglobul ins in agarosegel containing H-chain specif ic ant iserum to therespect ive immunoglobul ins.

Results and DiscussionThe values of serum lgG and lgM as measured in

control, hypothyroid and hyperthyroid groups areshown in Table 1. Serum lgG was 8OO * 154.49mg/dl in controls. The corresponding value inhyperthyroid pat ients was 127O t 160.21 mg/dlwhich was signi f icant ly higher than controls bu

remained within the normal physiological range.

lncrease in serum ISG has been observed in

hyperthyroid pat ients of Grave's disease, due to

the presence of ant ibodies of lgG classT. Thepresent study also included cases of Grave's

disease and the elevat ion of lgG could be due to

the presence of these antibodies.Serum lgM was 190 t 25,28 mg/dl in controlsand 177 t 16.02 mg/dl in hyperthyroid pat ients

which were not significantly different incomparison. Serum lgM was 92.7 t 20.03 mg/dlin hypothyroid pat ients which was signi f icant ly

18

lower than controls but st i l l i t wi thin the normalbiological range.Hypothyroid pat ients had serum lgGconcentrat ion of 446 t 38.28 mg/dl which wassignif icant ly lower than the control and sl ight lyless than the normal l imit . lgG part ic ipates inmost immunological react ions such ascomplement f ixat ion, precipi tat ion andneutral isat ion of toxins and viruses. i t b indsmicroorganisms, enhancing their phagocytosis,and serves as a general purpose ant ibody,protect ing,against the infect ive agents which areact ive in blood and t issues. Decrease in serumlgG, therefore, increases the r isk of infect ions.The exact mechanism by which thyroid hormonesmight inf luence lymphocyte funct ion and humoralimmunity is not known. The cel lular s i te of act ionof thyroid hormones is within the nucleus andthey inf luence a number of metabol ic processes,

stimulate calorigenesis and affect protein

synthesis, by their action on a variety ofenzymesE. Human lymphocytes possess nuclearreceptors for thyroid hormones through which

they possibly inf luence the synthesis of

immunoglobul inss. Other mechanisms might be

involved for the depression of humoral immunity

observed in hypothyroidism which have yet to be

establ ished.

AcknowledgementI wish to express my sincere thanks to Dr. P.C.

Ray, Dept. of Biochemistry, Maulana Azad

Medical Col lege, Delhi for hia valuable

suggest ions and advice.

Table 1. serum immunoglobul ins ( lgM and lgG) in hypo-, hyper thyroid pat ients bnd controls.

No. of cases lgG (mg/dl)

Mean + SD

lgM (mg/dl)

Mean + SD

ControlHypothyroidHyperthyroid

202020

8OO t 1 54.49446t38.28*

127Ot. l60.21 *

19Ot '25.2892.7 t2O.O3*177 + 16.02

Level of s igni f icance when compared to the controls *p <O.OOl '

H. J. Med. Biochem., 2OO1, 511&21

Referencesl . Bfalock, J.E. (19891 Physiol.8ev.,6911l:1 6. Tsai, J.C. and Samuels, H.H. 119741 J. Clin.

Endocrin. Metab.,51 : 1O6.

2. Bachman, S.E. and Mashaly, M. (19891 lmmun.

11 :203. 7. Mariotti, S, Chiovato, L, Vitt i, P. Marcocci, C.,

Fenzi, G.F., Delprate, G.F, Tiri, A. Romagnani, S.,

3. Starling, K.R. and Weese, J.L. 119851 J. Surg. Ricci, M. and Pinchera, A. 119891 Clin. lmmun.

8es.,39 :413 lmmunpath., 5O : 573.

4. Fabrisi N., Mocchegiani, Ei Mariottl, S., Pacini, F. 8. Tsai, M.J. and Malley, B.W. (1 9941. Ann. Rev.

and Pinchera, A. (19861. J. Clin. Endocdn. Metab.' Biochem.,63:451.

62111 z 474,

9. Relchlin. S. (19951. Physiol. Rev.,75l1l z 2O1.

5. Schoenfeld, P.S., Myers, J.W., Myers, L. and La

Rocque J.C. 119951 Southem Med. J., 88 : 374.

t9

lnherited Metabolic Disorders:Approach to Diagnosis by Biochemical Methods

Rita ChristopherDepartment of Neurochemistry, National Institute of Mentat Health and Neuro Sciences,

Bangalore 560029

AbstractOver the last two decades there has been a dramatic increase in our understanding of the molecularbasis of many of the inherited metabolic disorders. The precise diagnosis of these disorders, whichis a challenge to the physician, can be best accomplished by biochemical methods. Screening ofclinically selected pateints with simple chemical urine tests and routine blood chemistryinvestigations followed by measurement of specific metabolites and assay of the relevant enzymesconfirm the diagnosis in most cases. Biochemical diagnosis of inherited neurometabolic disorders israpid and confirmatory and therefore aids in treatment and further prevention of these raredisorders. Evaluation of inherited metabolic disorder in 8685 patients by biochemical analysis wasdone and reported here .

IntroductionThe study of inheri ted metabol ic diseases, whichwas ini t iated by Sir Archibald Garrod at the turnof this century, has grown at a dizzy pace,part icular ly dur ing the last two decades. Most ofthese disorders impair the funct ion of the nervoussystem, probably, because at birth the brain isimmature and therefore susceptible to chemicalderangements at many stages during itsdevelopmentr. The inherited metabolic diseasesinclude a number of individual ly tate butimportant disorders affect ing the metabol ism ofamino acids, complex l ip ids,

mucopolysaccharides, carbohydrates, fatty acids,purines and pyr imidines. These disorders havealso been known col lect ively as inborn errors efmetabol ism. Considerable progress in post andprenatal diagnosis has opened wide perspect ives,

especial ly for the prevent ion of these disorders,many of which are untreatable.An accurate, conf irmed diagnosis is important asthe foundat ion for any possible treatment. Earlydiagnosis helps in early intervent ion throughdietary manipulat ions, v i tamins or drugs.

Diagnot ic accuracy is also essent ial so thatparents may be informed of the prognosisalso of the genet ic impl icat ions in highfamil ies,The diagnosis of the inheri ted medisorders depends on a high index ofsuspicion and access to expert laborservices, part icular ly biochemical analysis.under ly ing defect can be invest igated at thr

di f ferent levels : the metabol i te level , the enlevel , and the DNA level .

Indicat ions for biochemical screeningSelect ion for the r ight biochemical screeningthe most crucial step in the diagnost ic proFor a c l in ic ian the problem of recogni t ion ofdisorders, especial ly in a neonate,around the non-specif ic i ty of the cl in ical s igand symptoms. Symptoms may be acuneonatal or develop slowly : they may alpresent suddenly at a later age, somet imes

intermit tent. Rarely, even a complexapparent ly incoherent, nonspeci f ic abnormal i t imay be observed.

Neonates wi th metabol ic disorders usual lv

normal at b i r th, however, s igns and symp

lnd. J. Med. Biochem.,20O1,5(1&2)

such as lethargy, poor sucking and feeding,convulsions and vomit ing may develop as earlyas a few hours after bir th. Lethargy, poor

feeding, convulsions and coma may also be seenin infants with hypoglycemia or hypocalcemia.Response to intravenous glucose or calciumusual ly establ ishes the diagnosis. Any or al l ofthese symptoms may also be present in a chi ldwho suffers from infect ion during the newbornperiod. The unexpected and relent lessdeterioration of a full-term baby after a normalini t ia l per iod is the most important s ign of thepresence of organic acidurias, urea cycle defectsand other aminoacidopathies or sugarintolerances l ike galactosemia. In fat ty acidoxidat ion defects, mitochondrial respiratory chaindisorders, glycogenoses and disorders ofgluconeogenesis the most frequent symptoms aregeneral ized hypotonia, rapidly progressive

neurologic deter iorat ion associated with

hypertrophic cardiomyopathy or malformations.The presence of hypoglycemia, hepatomegaly

and hepat ic dysf unct ion is suggest ive ofgluconeogenesis defects.Some disorders exhibi t mi lder var iant forms thathave a more insidious onset and may present in

late chi ldhood, adolescence or even adulthood.

Recurrent attacks of coma, vomitting and ataxia

may occur with progressive developmental delay

and abnormal behavior in a chi ld. Cl inical

symptoms of the lysosomal storage disorders areprogressive, permanent and independent of

intercurrent events. Visceral s igns such as

hepatosplenomegaly, dysmorphic f acies and

skeletal abnormal i t ies appear along withprogressive neurological deter iorat ion in most of

the lysosomal disorders. ln adults,

neurodegenerat ive symptoms such as psychosis,

dementia, ataxia, seizures, dystonia and

peripheral neuropathy can be reveal ing signs of

var iant forms of inheri ted metabol ic disorders.

The biochemical tests carr ied out on a pat ient

with a suspected inheri ted neurometabol ic

21

disease general ly fal l into four broad categories:i lgeneral metabol ic screening tests on blood andurine, i i )analysis of specif ic metabol i tes (amino

acids, organic acids etc. l , i i i )enzyme assays andivlDNA studies,

General metabolic screening testsThe f i rst approach to a biochemical evaluat ion isa mult icomponent analysis of the body f lu ids ofcl in ical ly selected pat ients, a procedure cal ledmetabol i te screening (Table 1 ) . Results of theprel iminary screening tests can often help tosuggest certain known groups of metabol icdisorders. Lysosomal disorders, peroxisomal

disorders, porphyrias, and glycogen storagedisorders are often suspected mainly on cl in icalgrounds and the pert inent diagnost ic tests can beperformed,

Urine screening tests : Several screening testscan be done on the ur ine. They are simple toperform and indicate further tests for rapidlaboratory diagnosis. However, ur ine screeningtests often give f alse posit ive react ions andtherefore the diagnosis should be conf irmed bythe quant i tat ion of the compounds of interest orenzyme assays.Unusual odors, i f present, can be detected in theurine. Maple syrup or burnt s igar- l ike odor is

character ist ic of maple syrup ur ine disease(MSUD). Acidi f icat ion of ur ine wi l l intensi fy theodor. lsovaler ic acid and isovalerylglycine, which

is excreted in pat ients with isovaler ic acidemia

have a character ist ic sweaty feet odor. Musty

odor has been used to descr ibe ur ine frompatients with phenylketonuria, and f ishy odor is

associated with tr imethylaminuria. Urine from

patients with p-methylcrotonic aciduria has a

odor simi lar to a tomcat 's ur ine. The f err ic

chlor ide test is usef ul for detect ion of

phenylketonuria (PKU).

Although a color react ion to f err ic chlor ide

reagent has been reported with MSUD and

hist idinemia, they are inconsistent and unrel iable.

Ind. J. Med. Biochem., 2OO1, 5(1&2)

Simi lar ly, ferr ic chlor ide test is unrel iable as adiagnost ic test for PKU during the f i rst few

weeks of l i fe and in pat ients who are part ial ly

treated.The dini trophenylhydrazine (DNPHI test whichdetects cr-ketoacids, is most usef ul in thediagnosis of MSUD and other organic acidurias.

This test is also a complement to ferr ic chlor idetest in the diagnosis of PKU. Commercial lyavailable reagent strips can be used to detectketone bodies in the ur ine. These str ips are mostsensitive for acetoacetic acid but can also react

with acetone and butanone. The cyanide

nitroprusside test detects any compound

containing sul f hydryl group and f inds i ts

appl icat ion in screening for homocyst inur ia,cyst inur ia, gluthathionuria, and P-mercaptolactate-cysteine disul f idur ia.

Reducing substances in the ur ine can be detected

by Benedicts test. The common substances

ident i f ied by this test are glucose, galactose,

f ructose, lactose, pentoses and homogentistic

acid. False posit ive results occur with nucleic

acids, polyphosphates, excessive epithel ial

excret ion and connect ive t issue destruct ion.

Urine screening for amino acid metabolic

disorders can be performed by qualitative

chromatographic techniques l ike paper or

thin-layer chromatography.During pregnancy hist idine excret ion is increased.

Thin- layer chromatography is also the most

suitable test for identifying the reducing sugarspresent in ur ine. Galactose, fructose or lactose

excreted in urine due to defective metabolism of

these sugars can be identified by this test.

Blood screening testsBlood glucose : Hypoglycemia is a prominent

feature of glycogen storage disorders Types I and

l l l as wel l as def ects of the gluconeogenic

pathway. Secondary hypoglycemia is seen in

MSUD, organic acidemias and disorders of fat ty

acid oxidat ion. In absence of hepatomegaly and

22

ketoacidosis, hypoglycemia suggests thepresence of organic acidur ias, ketolyt ic defects,

late-onset MSUD, and glycerol k inase

def ic iencies. ln inborn errors of ketogenesis and

f atty acid oxidation thero is hypoketotic

hypoglycemia.

Blood pH : Acidosis is a major metabolic

expression in several inher i ted neurometabol ic

diseases and therefore blood pH should be

assessed lor evidence of metabolic acidosis.

Disorders caused by excessive H* product ion as

a resul t of inborn errors of metabol ism,

col lect ively cal led organic acidemias, are largely

caused by impaired metabol ism of the

branched-chain amino acids or pyruvate. Pat ients

with organic acidemias present a few days af ter

bir th wi th unexplained severe metabol ic acidosis,

lethargy, feeding problems, vomit t ing and

tachypnoea. Lesser def ic iencies of ten manifest as

an episodic course of recurrent encephalopathy

that includes vomit ing, a l tered consciousness,

and ataxia. The disorders that cause episodic

metabol ic acidosis are : Pyruvate dehydlogenase(PDH) complex disorders, mult ip le carboxylase

def ic iency, intermit tent MSUD, propionic

acidemia, methylmalonic acidemia and fat ty acid

acyl-CoA dehydrogenase defects.

Blood lactate : Many inher i ted metabol ic

disorders ref lect their under ly ing enzymatic defect

in an abnormal pattern of lactate and pyruvate.

Ketosis is present in most of the pl imary lact ic

acidemias but is absent in acidosis secondary to

t issue hypoxia. In defects of g luconeogenesis due

to def ic iencies of g lucose-6-phosphatase and

fructose-1,6-biphosphatase, the lact ic acidemia is

usual ly permanent and can be exacerbated by

fast ing and hypoglycemic episodes in af fected

chi ldren. Pat ients wi th glucose-6-phosphatase

def ic iency are short-statured and present wi th

hepatomegaly, nephromegaly, adiposi ty, lethargy,

seizures, epistaxis and xanthomata accompanied

by hypoglycemia, hypertr ig lycer idemia,

hypercholesterolemia, hyperur icemia and ketosis-

Ind. J. Med. Biochem., 2O01, 5(1&2)

Fructose 1,6-biphosphatase def ic iency isassociated with lethargy, i r r i tabi l i ty,hepatomegaly seizures, hypoglycemia andketosis. Profound hyperlact ic acidemia occuringin pyruvate carboxylase def ic iency is associatedwirh a high lactate/pyruvate (L/P) rat io,post-prandial ketonemia, ci trul l inemia,hyperammonemia and hepat ic dysfunct ion. Inpyruvate dehydrogenase def ic iency, hyperlact icacidemia is permanent, increasing after meals anddecreasing during fast ing. The L/P rat io is normalor low. Organic acidemias, c i t rul l inemia, and fattyacid oxidat ion defects lead to lact ic acidemia as asecondary phenomenon.

Ketone bodies : High levels of the ketone bodies,acetoactate and p-hydroxybutyrate, are presentin blood in many inheri ted metabol ic diseasesincluding :( i ) Disorders of branched-chain amino acidmetabol ism in maple syrup ur ine disease,methylmalonic, propionic and isovaler icacidemias;( i i ) Congenital lact ic acidosis, such as mult iplecarboxylase and pyruvate carboxylasedef ic iencies, and respiratory chain disorders.( i i i ) defects in enzymes of gluconeogenesis suchas glucose 6-phosphatase, fructose1,6-biphosphatase and glycogen synthase;

{ iv) Ketolyt ic defects l ike 2-oxoacidCoA thiolasedef ic iencies; The presence of ketonuria is alwaysabnormal in neonates.A decreased production of ketone bodies isobserved in the inborn errors of fat ty acid andketone body metabol ism2. They include :i Mult ip le mitocl- iondrial acyl-CoA dehydrogenasedef ic iencies;i i lnheri ted disorders specif ical ly af fect ingmitochondrial long chain fat ty acid oxidat ion l ikecarni t ine palmitoyl t ransferase I and l ldef ic iencies;i i i Medium chain acyl-CoA dehydrogenasedef ic iency;iv Hydroxymethylglutaryl-CoA lyase def ic iency;

23

Blood ammonia : Hyperammonemia is abiochemical indicator of abnormal i ty of ni t rogenhomeostasis. The most common cause ofhyperammonemia may be related to abnormal i t iesin l iver metabol ism al though a var iety of inheri teddisorders may be responsible. Urea cycledisorders may present in the neonatal per iodwith non-specif ic symptoms such as lethargy,i rr i tabi l i ty and feeding di f f icul t ies after a var iablesymptom-free interval3. The blood ammonia r isesto levels higher than 4OOrrM. Respiratoryalkalosis and moderate hyperlact ic acidemia arefreguently observed. Less severe enzyme defectsmay present after the neonatal per iod withdevelopmental delay, f ai lure to thr ive orpersistent vomit ing. Older chi ldren have episodicor f luctuat ing i l lness with non-specif ic symptomssuch as behavioural abnormal i t ies lethargy,confusion, headache, ataxia or focal neurologicalsigns. In al l pat ients with hyperammonemia, i t isessent ial to measure plasma amino acids sincethey may be diagnost ic.Coagulat ion f actor deplet ion, elevated serumtransaminases, and hepatomegaly are f requentpart icular ly in argininosuccinic acid lyasedef ic iency. An important c lue to separate ureacycle defects f rom organic acidurias can beident i f ied by organic acid analysis in the ur ine. lnthe tr ip le H syndrome (HHH), hyperammonemia,hyperorni thinemia, and homocitrul l inemia arepresent. Fatty acid oxidat ion disorders which canalso present with hyperammonemia areassociated with hypoglycemia, hepat icdysfunct ion, muscular and cardiac symptoms andsudden infant deatho.

Liver funct ion tests : Jaundice and other evidenceof hepat ic dysfunct ion is observed in tyrosinemiatype l , galactosemia, hereditary f ructoseintolerence, and Wilson's disease. Liverdysf unct ion accompained by neurologic signs,severe hypotonia, developmental delay,cardiomyopathy and hyperlact ic acidemia is seen

Ind. J. Med. Biochem., 2001, 5(1&2)

in glycogenosis type lV and respiratory chaindisorders. When there is cholestat ic jaundice inthe neonatal per iod, in addit ion to crr - ant i t rypsindef ic iency which is the most f requent cause,Niemann -Pick type C, and peroxisomal disordersl ike Zel lwegers syndrome, and infant i le Refsumdisease should be considered.

Specif ic metabol i te assaysQuanti tat ion of abnormal amounts of normalmetabol i tes in the body f lu ids of a pat ient

suspected of having an inheri ted metabol icdisorder can conf irm the diagnosis in manycases.These metabol i tes mainly include aminoacids, organic acids, long-chain fat ty acids,mucopolysaccharides, ol igosaccharides, pur ines

and pyr imidines.

Amino acid analysis : A quantitative analysis of

the amino acid acids in blood and ur ine isnecessary for the confirmation of disorders ofamino acid .metabol ism. lon-exchangechromatography coupled with post-column

ninhydrin derivat izat ion is the technique mostwidely used f or rel iable separat ion andquant i tat ive determinat ion of amino acids5. lnrecent t imes, with the advent of high-pressurel iquid chromatography (HPLC), amino acid prof i le

in biological f lu ids is often based on reversephase HPLC after precolumn derivat izat ion with

o-phthaldialdehyde or some other f luorescent

compounds6. Disorders of amino acid metabol ism

show a charater ist ic plasma and ur ine amino acidprof i le which is diagnost ic. One of the most

common aminoacidopathies is phenylketonul ia,

which is marked by the massive excret ion ofphenylalanine and i ts metabol i tesT.Most of the disorders of the urea cycle,

homocyst inur ia and tyrosinemias show a

character ist ic elevat ion of an amino acid.

Organic acid analysis : The analysis of organicacids by gas chromatography mass spectrometry

24

(GCMS) is of value in the diagnosis of a widerange of disorders of amino acid and organic acidmetabol ism and f at ty acid oxidat ion def ects8.Some disorders, such as propionic acidemia,methylmalonic acidemia, isovaler ic acidemia, and3-hydroxy-3-methylglutar ic aciduria can berel iably diagnosed from organic acid excret ionsbecause of the consistent high elevat ions of thecharacter ist ic acids. In classic MSUD the elevatedmetabol i tes are usual ly 2-oxoisocaproic acid,2-oxo-3-methylvaler ic acid, and

2-hydroxyisovaler ic acid. An elevated level ofglutar ic acid is the major abnormal i ty of glutar icaciduria type l , but a smal l elevat ion of3-hydroxyglutar ic acid is unique to this disorderand aids in dist inguishing i t f rom mult ipleacyl-CoA dehydrogenase def ic iency in which2-hydroxyglutar ic acid is usual ly elevateds.Measurement of saturated unbranched fatty acidswith a carbon chain length ol 22 or greater, thevery long chain fat ty acids (VLCFAI, in plasma,. is

necessary for the ident i f icat ion of peroxisomal

disorders l ike Zel lwegers syndrome,adrenoleukodystrophy, and inf ant i le Ref sumdisease. The individual peroxisomal disorders canbe f urther ident i f ied by measuring othermetabol i tes l ike plasmalogens, bi le acids,phytanic acid etcro.

Urinary glycosaminoglycans (GAGs) and others :

Analysis of the ur inary GAGs in pat ients with aposit ive spot test f or mucopolysaccharidur ia

f aci l i tates the di f f erent ial diagnosis of

mucopolysaccharidoses by demonstrat ing the

biochemical defect and point ing to the probable

enzyme defect. Pat ients with Hurler (MPS lH),

Scheie (MPS lS) and Hunter (MPS l l ) excrete

excess dermatan and heparan sulf ate. Keratan

sulfate and chondroi t in sul fate are excreted in the

urine of pat ients with Morquio disease (MPS lV)

and dermatan sulfate in pat ients with MaroteauxLamy disease (MPS Vl). Pat ients with Sly

syndrome (MPS Vl l) have been found to excrete

rd. J. Med. Biochem., 2001, 5(1&2)

excess chondroi t in sul fate and dermatan sul fate.

Serum transf err in, copper and ceruloplasmin

determinat ions should be carr ied out in pat ients

, ' , , i th general ized parkinsonian r ig id i ty wi th

cysarthr ia, dysphagia or a postural or intent ion

iremor, psychiatr ic abnormal i t ies or hepat ic s igns

io detect cases of Wi lson's diseaserr . The

measurement of ur inary orotate and carni t ine in

the plasma and ur ine is indicated in pat ients who

f ai l to thr ive, have hypotonia, chronic muscle

rveakness and intermit tent episodes of weakness

or encephalopathy.

Enzymes studiesA number of storage disorders, part icularlylysosomal storage diseases, are not expressed atthe metabol i te level . Abnormal products do not

appear in the body f lu ids at a l l or have not yet

been detected as a consequence of both low

concentrat ion, compl icated structure and other

technical d i f f icul t ies. In certain cases, detect ion

of the abnormal metabol i tes may be labor ious and

t ime-consuming. For these disorders, screening at

the enzyme level is necessary. In disorders l ike

the mucopolysacchar idoses, enzymes assays are

required to determine the speci f ic subtype,

The act iv i ty of these enzymes can be measured

in easi ly obtainable or accessible t issue samples

such as plasma, leukocytes or cul tured skin

f ibroblasts. Table 2 . A few lysosomal disorders

are caused by lack of a protein cofactor (eg.

sphingol ip id act ivator protein) or mentbrane

transport proteins. Hence, pat ients who have

normal enzyme values and yet have cl in ical

f indings suggest ive of a lysosomal disorder

should be considered for addi t ional studiest2. The

f inal conf i rmatory diagnosis of most other

neurometabol ic disorders st i l l rests on

demonstrat ion of the abnormal enzyme act iv i ty.

For example, in the urea cycle disorders, ASLD,

ASD and arginase def ic iency can be conf i rmed by

assay of the enzyme act iv i ty in red cel ls,

leukocytes or skin f ibroblasts.

25

DNA diagnosis : Diagnosis at the DNA level bymeans of restr ict ion f ragment lengthpolymorphism (RFLP) or wi th speci f ic probes

provides a solut ion for the diagnosis, especial lyprenatal d iagnosis, where enzyme def ic iencies are

not expressed in amniot ic f lu id and cel ls or in

chor ionic t issue. Another major appl icat ion ofDNA analysis is genotype/phenotype correlat ion,presymptomat ic diagnosis and heterozygote

detect ion.

Inher i ted Metabol ic disorders at NIMHANS

During the last f ive years 8685 pat ients wi th

signs and symptoms suggest ive of inher i ted

metabol ic disorders were referred to the Cl in ical

Biochemistry Uni t at NIMHANS for evaluat ion.

In i t ia l screening tests were done on blood and

ur ine samples of these pat ients af ter which thequant i f icat ion of the speci f ic metabol i tes was

carr ied out based on the provis ional c l in ical

diagnosis. In cases of suspected amino acid

disorders the total amino acid concentrat ions

in blood and ur ine were est imated and the

indiv idual amino acids were quant i f ied by

reverse-phase HPLC after precolur.nn

der ivat izat ion wi th o-phthaldialdehyde. Reducing

substances present in the ur ine were ident i f ied by

thin- layer chromatography of sugars. Assays of

the appropr iate lysosomal enzymes were carr ied

out in the serum or leukocytes whenever

sphingol ip idoses were suspected. Using these

biochemical methods, 47 pat ients wi th var ious

disorders of amino acid metabol ism, 4 cases with

disorders of carbohydrate metabol ism, 148 cases

of the mucopolysacchar idoses, 58 cases of

sphingol ip idoses and 3 mucol ip idoses were

detected (Table 3). Al l pat ients were advised

appropr iate t reatment wherever f easible and

f ami ly members given the necessary genet ic

counsel l ing. In most of the cases biochemical

analysis provided a conf i rmed diagnosis obviat ing

the need for more invasive diagnost ic procedures

l ike l iver or brain biopsies.

Ind. J. Med. Biochem., 2OO1, 5(1&21

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2, Duran M. and Waldman, S,K. {19871 Enzyme,38 z

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3. Brusi lou, S.W. and Horwich, A. The

Metabolic and Molecular Basis of lnherited

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Buttersworth-Heinemann, Oxford, p,1 45.

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. Odor

. Ferric chloride test

. Dinitrophenylhydrazine (DNPH) test for

cr-ketoacids

.Cyanide nitroprusside test for sulphydryl

compounds

.Benedicts tests for reducing substances

. Strip test for ketone bodies

. Spot tests for mucopolysaccharidur ia

. Chromatography (Paper/Thin layer) for amino

26

Nyhan, W.L. and Hass, R. (19971 In : The

Molecular and Genetic Sasrb of Neurological

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Barchi RL (edsl Buttersworth-Heinemann, Boston,p.1 1 29.

Sweeten, L. (19911. ln : Techniques in Diagnostic

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29.:821.

Stibler, H., Jaeker, J. and Kristiansson, B. (19911,

Acta Paediatr. Scand.,375 lsuppl.l : 21.

12. Wenger, D.A. and Wil l iams, C. 11991) In :

Techniques in Diagnostic Human Biochemical

Genetics : A Laborctory Manual, (Homes F.A.A.

Ed.l Wiley-Liss Inc., New York, p.587.

Blood

. Glucose

. pH and electrolytes

. Lactate

.Ammonia

. Ketone bodies (acetoacetate, p- hydroxybutyrate

. Liver function tests

. Chromatography (Thin layer) for amino acids.

7.

8.

9.

10.

11.

Table 1. lnitial Screening Tests for Inherited Metabolic Disorders.

lnd. J. Med. Biochem., 2001, 5(1&21

Disorder

G*r, gangl iosidosis

Gn, gangl iosidosis

Tay Sachs diseaseSandhoff diseaseAB variant

Metachromatic leukodystrophyAl l c l in ical typesSAP-1 def ic iency

Krabbe's diseaseFabry's diseaseNiemann-Pick disease

Typeb A & BTypes C

Gaucher 's diseaseTypes 1,2 & 3SAP-2 deficiency

Fucosidosis

a and p-Mannosidosis

Sial idosisMucol ipidosis l l & l l l

Multiple sulfatase deficiencyMPS I Hurler, ScheieMPS l l HunterMPS I I I

Sanf i l ippo ASanfilippo BSanfilippo CSanfilippo D

MPS IVMorquio AMorquio B

MPS Vl Maroteaux LamyMPS Vl l Sly Syndrome

Table 2. Specif ic tests indicated in var ious lysosomal disorders.

Diagnost ic Test

p-Galactosidase

p-Hexosaminidase AHexosaminidase A & BG", act ivator protein level

Arylsul fatase ASAP-1 level, sul fat ide loading testGalactocerebrosidaseor-Galactosidase A

Sphingomyel inaseCholesterol esterif ication

p-Glucosidase

SAP-2 level

a-L-f ucosidasecr-L-Mannosidase & p-L-Mannosidase

ct-Neuraminidasep-Galactosidasep-Hexosaminidase

Arylsul fatase A & Bcr,-L-lduronidaselduronate sul fatase

Heparan N-sulfatasecr-N-acetylglucosaminidaseAcetyl-CoA: glucosaminide acetyl t ransferaseN-acetylglucosamine 6-sulf atase

G alactose-6-sulf atasep-Galactosidase

Arylsul fatase Bp-Glucuronidase

P . .Plasma, L . .Leucocytes, F . .Fibroblasts

27

Tissue Used

P,L,F

P,L,FP,L,F

F

P,L,FF

L,FL,F

L,FL,F

L,FF

P,L,FP,L,F

L,FP,L,F

P,L,FP,L,F

F

L,FP,L,F

FL,F

L.FP,L,FP,L,FP,L,F

lnd. J. Med. Biochem., 2OO1, 5(1&21

Table 3. Inherited Metabolic Disorders Detected at NIMHANS on Screening 8685 Patients.

Disorders

Disorders of amino acid metabolismPhenylketonuria

AlkaptonuriaMaple syrup urine disease

Hyperonithinemia associated with gyrate atrophy of choroids and retinaTyroginemiaNon-kstotic hyperglycinemiaCitrul l inemiaHyperargininemiaHyperprolinemia, type IHomocystinuriaSulfite oxidase defect

The Sphingol ipldosesMetachromatic leu kodystrophyTay-Sach diseaseSandhoff diseaseJuvenile GM2 gangliosidosis

Multiple sulfatase deficiencyNiemann-Pick, type A

Disorders of carbohydrate metabolismGalactosemiaGlycogen storage disorder

MucopolysaccharidosesMucolipidoses

Sialidosis, type Il-cell diseasePseudo-Hurler polydystrophy

Disorders of purine metabolismLesch Nyhan syndrome

Disorders of porphyrin metabolismAcute intermittent porphyria

1110754222211

291310411

22

148

111

2

2

Ind. J. Med. Biochem., 2001, 5t1&21

Detection of ACTH Peptides in Breast Cancer Tissues

A. Ray*, A.K. Bahadur**, D. Jain**, S.L.D. Naik+, A.B. Mitra* and B.K. Sharma**lnstitute of Cytology and Preventive Oncology (lCMRl and *t Department of Radiotherapy, Maulana

Azad Medical Gol lege, New Delhi-110002.

AbstractAdrenocorticotropic hormone (ACTH), an anterior pituitary hormone, is processed from precursor

pro-opiomelanocortin (POMCI molecule along with other peptides like melanocyte-stimulatinghormone (MSHI, endorphins, etc. In the present study, ACTH peptides were detected in tissues of19.2o/o cases of breast cancer. Interestingly, a statistically significant correlation was noticedbetween the positivity of ACTH and the positive oestrogen receptor (ERl status. However, thepresence of ACTH peptides did not show any association with other parameters such asmonopausal status of the patients, stages of the disease, metastases to lymph nodes andprogesterone (PgRl receptor status. The findings of this study are significant in order toevaluate the precise steroid hormones related pathology of breast cancer.

29

IntroductionNeuroendocrine cells ate found in severaltumours. These cel ls produce variousneuropeptides which may be involved inautocrine/paracrine pathway of regulation ofgrowth, differentiation, secretion andneuroendocrine-immune interactionst. Tumourswhich produce such polypept ide hormones maycause paraneoplastic syndrome, for exampleectopic ACTH syndrome, syndrome ofinappropriate ant idiuret ic hormone (SIADH) dueto ADH or an ADH-Iike substance, calcitoninproduct ion by tumours, etc.Ectopic ACTH

syndrome is commonly associated with smal l-cel l

carcinoma of the lung but can also be found in a

variety of neoplasms l ike carcinoid tumours of

bronchus, thymus and gut, is let-cel l tumour ofpancreas,medul lary carcinoma of thyroid,pheochromocytoma, ovarian adenocarcinoma,etc2. ln i t ia l ly, i t was thought that in ectopic

ACTH syndrome, the tumour produced ACTH or

an ACTH-l ike substance which led to adrenal

hyperplasia and hypercort isol ism. Subsequent ly,

the predominant active molecules involved in the

syndrome have been shown to be precursors ofACTH, containing not only ACTH but also MSH,p-LPH, endorphins and enkephal ins.ACTH and its related peptides are secreted bybasophi l ic cel ls (cal led cort icotrophs) of anter iorpituitary. ACTH is a 39-amino acid peptideprocessed from a large precursor molecule,POMC. Within the cort icotroph, a single mRNAdirects the synthesis and processing of POMCinto smaller biologically active fragments whichinclude P - LPH, a-MSH, p- MSH, F - endorphinand the amino terminal f ragment of POMC. Thehypothalamic hormone CRH, a 41-amino acidpept ide, st imulates the secret ion of ACTH and

other products of i ts precursor molecule, POMC.

Breast carcinoma is a rare cause of ectopic ACTHsyndrome. However, Wigg et al3 descr ibed a

breast cancer case who presented with clinical

and biochemical features- of ectopic ACTH

syndrome ( in metastat ic disease). They observed

that immunostaining of the breast metastases for

ACTH was posit ive and in-si tu hybridizat ionrevealed POMC gene expression. Chobanian et ala

f ound a direct relat ionship between

Ind. J. Med. Biochem., 2001, 5(1&21

CD1 1b-posit ive cel ls and ACTH basal secret ion inmiddle-aged females with breast cancer. l t hasbeen suggested that the integrity ofneurohormonal routes is needed in addit ion to anintact immune system to elicit an effectiveimmune responses. The ACTH const i tutes animportant aspect of neurohormonal route2'6.Studies from various angles have indicated thatsteroid hormones are strongly associated withthe pathological process of breast cancer.Further, the ACTH has an important place in thehypothalamic-pituitary-adrenal system and itstimulates the initial phase of steroid hormonebiosynthgses ( i .e. , cholesterol to pregnenolone) inadrendl cortex. Therefore, the aim of the present

study was to detect the ACTH or ACTH-likepeptides directly in the tissues of female breastcancer which is dependent on steroid hormone.

Materials and MethodsThe present study was conducted on 26 cases ofinfiltrating duct carcinoma of the breast, which

were randomly selected from the patients

admitted in Lok Nayak Hospital (Maulana AzadMedical Col legel, New Delhi . As negat ive control ,5 cases of f ibroadenoma of the breast (benign

disease) were also included in this study.Ouestionnaires related with clinical and

epidemiological parameters were used tor allcases and controls. The' basic information on

socio-demographic aspects was collected,

Personal history, relevant family history,

obstetrical and gynaecological history were also

taken. Out of 26 cases of breast cancer, 1 3

pat ients were premenopausal and the rest 13

pat ients belonged to postmenopausal group.

lmmunochemical staining: The experiment was

carr ied out on 5pm thick formal in f ixed paraff in-

embedded t issue sect ion from breast tumours,

according to the method described by Ratnakar et

al7. The t issue sect ions were deparaff in ized by

washing with xylene/toluene solut ion and later

subjected to successive lower concentrat ions of

30

ethyl alcohol. Subsequent ly, they were washed inphosphate buffered sal ine (PBS, pH: 7.2l , . Tissuesect ions were incubated for 30 min with freshlyprepared 3% solut ion of hydrogen peroxide inmethanol to block endogenous peroxidaseact iv i ty, fol lowed by incubat ion in mouse normalserum. Then, opt imal ly di luted pr imary ant ibodyagainst ACTH (Sigma USA) was appl ied to t issuesect ion and incubated lor 60 min at roomtemperature in a humid chamber. Simi lar ly,monoclonal ant ibodies against ER (ER-DS ant igen,Amersham, UK) and PgR (Sigma USA) were usedto evaluate the receptor status of tumours.Afterwards, the sect ions were washed with PBSand then they were incubated with secondaryant ibody (ant i-mouse lgG-Fab specif ic) f or 60min. After washing in PBS (along with O.OO5%Tween 2Ol, the t issue sect ions were incubatedwith peroxidase-ant iperoxidase complex for 6Omin at room temoerature. The react ion wasvisual ized by f reshly prepared substratediaminobenzidine (DAB : O.1o/o solut ion in PBSwith 0.05% hydrogen peroxide). After thedevelopment of colour, the t issue sect ions werewashed with double dist i l led water and thencounterstained with Harr is haematoxyl in.Subsequent ly, the sect ions were dehydratedthrough graded alcohol and then mounted withDPX. Afterwards, the sl ides were assessed undermicroscope.Stat ist ical analysis : The data were analysed to

est imate the proport ion of posi t iv i ty in var iousgroups. Moreover, Fisher 's exact test of

signi f icance was appl ied f or comparing the. associat ion between di f lerent var iables.

ResultsIn this study age of the pat ients with breastcancer was 46.0 t 12.4 years. The mean age ofpremenopausal pat ients (n = 13) was 37,4 t 5,5years and mean age of postmenopausal pat ients(n = 13) was 54.7 x 11.2 years ' Number ofissues was 2.96 t 1.5 among breast cancerpat ients. Out of 26 breast cancer cases, 7

Ind. J, Med. Biochem., 2O01, 5(1&21

pat ients belonged to stage lV, 14 stage l l l and 4stage l l . Metastasis in lymph nodes was observedin 22 pat ients.In the present study, ACTH pept ides (Fig 1) weredetected in 5(19.2%l cases only. Out ot 26breast cancer cases, 9 134.60/o) showedimmunohistochemical posi t iv i ty for ER and 13(5Oo/o) cases were posit ive for PgR. ln this study,23.1o/o (3/131 postmenopausal cases and 15,4o/oQl13l premenopausal cases revealed theexpression of ACTH pept ides. On the other hand,60% (3/5) of ACTH posit ive cases belonged topostmenopausal group. Among cases with higherstages ( l l l and lV), 19olo l4l21l were posit ive forACTH ., whereas, 1 case (out of 5) of lowerstages (below l l l ) showed presence of ACTH. Onthe contrary, 8Oo/o (4/5) ACTH positive casesbelonged to stage l l l . Out of 22 cases with lymphnode metastases, 4 l18.2ohl cases also revealedthe posit iv i ty for ACTH, whi le 1 case (out of 4having no involvement of lymph nodel withoutmetastasis was positive for ACTH, Further, itwas noticed that 8O7o l4l5l ot ACTH - positivecases showed lymph node metastases (Table 1).lnterestingly, in the present study, a significantcorrelat ion was observed between the product ionof ACTH pept ides by tumours and their ER status(pcO.OSl. Out of 9 ER posit ive cases, 44.4o/ocases were posit ive for ACTH and out of 17 ERnegative cases, 5,9% (only 1 case) showedpositivity for ACTH. Alternatively, 8Oo/o l4l5l otACTH producing tumours also revealed theexpression of ER. However, unl ike ER, PgR didnot show any statistically significant relation withthe expression of ACTH pept ides. Out of 13 PgR

- positive breast cancer cases, only 1 5.4o/o l2l13lcases were ACTH producing tumours. Simi lar ly,amongst PgR negat ive cases, only 23.1o/o (3113l,

cases were positive for ACTH. Further, out of 5ACTH - positive tumours, 2l40o/ol cases wereposit ive for PgR (Table 11.

DiscussionHormone-dependant or endocrine-related cancers

have an important place in the f ie ld of oncology.

31

Cancers of var ious organs ( l ike endometr ialcancers, breast cancer and prostatic cancere) inour body may show a relat ion with hormonalfactors Notwithstanding, the epidemiologicalevidence strongly supports the theory thatendogenous oestrogens play a crucial role inbreast carcinogenesis. Further, animal studieshave repeatedly demonstrated that oestrogenscan induce and promote mammary tumours inrodents. In human, nul l ipar i ty, menarche at ear lyage f irst f ull-term pregnancy at late age andmenopause at late age have been shown, formany years, to be associated with an increasedrisk of breast cancerto. The pr imary source ofoestrogens in postmenopausal women is from theconversion of androstenedione to oestrone inperipheral t issues, including adipose t issue, thuspostmenopausal obesity increases the r isk ofbreast cancer through increased production ofoestrogens. The conversion of androgens tooestrogens is catalysed by the enzyme complexaromatase (aromatizat ionl . l t has beendenostrated that certain breast carcinomas havethe ability to produce oestrogen throughintratumoural aromatasel r .

Al l mammalian steroid hormones are formed fromcholesterol v ia pregnenolone through a ser ies ofreact ions that occur in the adrenal cel l . ACTH isresponsible for the synthesis and release of theseadrenal steroids, v iz. glucocort icoids,

mineralocorticoids and sex-hormones.Glucocort icoids play a role in the regulat ion ofCYP19 gene transcr ipt ion in breast adipose

t issuer2. The biosynthesis of oestrogen f fom C19steroidb is catalyzed by a specif ic f orm of

cytochrome P450, namely aromatase cytochrome

P450, the product of the CYP1 9 gene. Further,

oestrogen produced by stromal cel ls surroundingthe tumour acts to st imulate the product ion ofgrowth f actors and cytokines by the tumour

cel ls. These or other f actors may act on the

surrounding mesenchymal cel l in a paracr ine

fashion to st imulate aromatase expression in the

lnd. J. Med. Biochem., 20O1, 5(1&2)

presence of g lucocort icoidsr3'ra. Zarghami et a l rs

observed that 30-40% of female breast tumours

produce prostate-speci f ic ant igen (PSA) and that

PSA product ion is associated with the presence

of oestrogen and progesterone receptors. The

breast carcinoma cel l l ine T47- D produces PSA

when st imulated by androgens, progest ins and

glucocort icoids or mineralocort icoids (but not

oestrogens). L ike PSA, human glandular Kal l ikrein

(hK2) is also produced in breast cancer cel l l ine

T47 -D af ter steroid st imulat iont6 ' Further,

Diamandis and YurT demonstrated that the gene

expression and protein product ion of PSA in

non-prostat ic t issue are under the regulat ion of

steroid hormones via their receptors. Androgens,

glucocort icoids and progest ins up-regulate the

PSA gene expression. On the other hand, matr ix

metal loproteinase (MMP) was one of the f i rst

proteinases found to be associated with cancer;

and glucocort icoids inhibi t the MMP synthesis.

Therefore, g lucocort icoids, MMPs and disrupt ion

of the basement membrane may play a key role in

induct ion and progression of breast cancerts.

Overal l , wi th in the environment of steroid

hormones, presence. of ACTH pept ides in breast

cancer t issue, perhaps, indicates a s igni f icant

role.

According to Chat ikhine et a l te, neuropept ide

expression is not cancer-speci f ic , i t could be

cancer related. Neuropept ide expression may

ref lect the host response to cancer cel ls and not

only the cancer cell activity. Heiny et al2o

observed a s igni f icant correlat ion between NK-

and T-cel l act iv i ty and p-endorphin plasma level

among breast cancer pat ients. They ant ic ipated

an obvious correlat ion between immune system

and the neuroendocr ine system, which might

gain therapeut ical re levance. On the other hand,

van der Pompe et a l2t found that breast cancer

pat ients had signi f icant elevat ions in basal

cort isol levels compared to controls, and

metastat ic breast cancer pat ients had higher

cort isol levels than ear ly stage breast cancer

32

pat ients. So, they assumed that breast cancer is

associated with a hyperact ive adrenal g land.

Further, several studies have revealed thatwomen with breast cancer have higher bloodlevels of testosterone or androgensrt '22. The

source of excess androgens arnong breast cancerpat ients may be adrenal g lano. Disturbances in

extra-adrenal regulators l ike ACTH or ectopic

source of ACTH may lead to excessive adrenal

sex-steroid product ion". In our ear l ier study,immunoposi t iv i ty for ACTH ' , . ras found in 15%

cases of inf i l t rat ing duct carc,nc: : ra of the breast,

and also, a posi t ive asscc,e: : - f ,e i , , ' , 'een the

immunoposi t iv i ty of ACTH a^: l i . , ' ,as not iced23.

Simi lar ly, in the preseni s: , : . . ' .e ocserved a

signi f icant relat ionship Dei , ' , e:- - - - roposi t iv i tyof ACTH and ER. Out of 5 cas=s . ' , r , ,ch showed

immunostaining for ACir : : : : :es, 4(80%)

cases were also posi t r ' , 'e 'c ' ! i ^ : ie present

study, 19,2Vo (5126t cases ' : . :a ej posi t ive

immunostaining for ACir - - : : :esence of

ACTH or ACTH-l ike Dei : :€s ^ : :east cancer

t issues and their re lat iors- : . ' . : - ER perhaps

indicate a compl icated s:e ' : : e- . -onment. An

elaborat ive study compr s - : . ' . a ' ,cus steroid

hormone related f aclors e- = .1- et pert inent

factors in relat ion r ,vr tn A 3-- .a- ce able to

evaluate the precise neL:3e- ==." : cnaracter of

breast cancer as wel l as :s ---- ,c logical and

prognost ic s igni f icance.

Fig. 1: Shows posi t ive immunochemical staining

for ACTH neuropept ides rn breast cancer t issue

(x400).

Ind. J. Med. Biochem., 2OO1, 5(1&2)

Table 1. Presence of ACTH neuropeptides and their relationship with menopausal status, stage of thedisease, metastasis in lymph node and receptor status (ER and PgR) in breast cancer.

ACTH Peptides

Posit ive (n = 5) Negat ive (n = 21 l

Menopausal status Post menopausal(n = 131

Pre menopausal(n = 131

3/5(60%l3l '13123.1o/ol

215(4QYol2113 l15.4Vol

10121 (47.60/ol

1O/1 3 (76.9o/ol

11121 (52.4o/ol

11 113 184.60/0lP=0.5

Stages l l l & lV (n=21)

Below l l l (n=51

415 lSQo/ol4121 (19%ol

115 l2oo/ol

17 121 l81o/ol

4121 119o/ol4t5 t80%lP = 0.69

Lymph node status Positive ln=221

Negative (n = 4l

415 lBOo/ol4t22118.20h1

115 (2oo/ol

1t4 l250hl

18121 lB5.7o/ol' t8t22 (81.8%l

3121 (14.3Yo1

314l7i%olP =0.6

ER Posit ive (n = 9l

Negat ive (n = 171

415 lSoo/ol419 144.4o/ol

115 l2Oo/ol't t17 l5.9%ol

5t21 123.8%l5/9 (55.6%t

16121 |.76.20/ol

16117 194.1o/ol

P<0.O5*

PgR Posit ive (n = 13)

Negative (n = 131

215 (4OYol

2113 115.4o/ol

3/5 (607ol

3113 Q3.1Yol

11121 152.4o/ol11113 (84.6olol

10121 147.60/ol1O/13 | '76.9o/olP=0.5

* Signif icant

lnd. J. Med. Biochem., 20O1, 5(1&2)

References1. diSant 'agnese, P.A. and Cockett , A.T.K. (19941. 12, Agarwal, V.R., Bulun, S.E., Lei tch, M., Robr ich, B.

J.Urol., 152 : 1927. and Simpson. E.R. (19961. J. Clin. Endocrinol.Metab.,81 : 3843.

2. Ray, A. (1998). A.M.B.I. Bull. lDelhi), 1 :25.13. Schmidt, M. and Loffler, c. (19941. Eur. J. Cell.

3. Wigg, S.J. , Ehr l ich, A.R. and Ful ler , P.J. (1999). Biol . ,63:96.

Clin. Endocrinol. (Oxf.), 50 : 675.14. Cr ichton, M.8. , Nichols, J,E.. Zhao, Y., Bulun,

4, Chobanian, N.S., Kushl inski i , N.E., Bassalyk, L.S., S.E. and Simpson, E.R. {1996}. Mol. Cel l .Kuzmina, 2.V., Gershtein, E.S., Saveleva, E.V., Endocr inol . , 118:215,

Baryshnikov, A.l. and Kadagidze, Z.c. (1 9941. l in,

Med. lMosk),72 : 37. 15. Zarghami, N., Grass, L. and Diam'andis, E.P.(19971. Br. J. Cancer. ,75 :579.

5. M-araveyas, A., Hrouda, D. and Dalgleish, A.G.(19981. ln, Molecular Aspects of Cancer and its 16. Hsieh, M.L., Charlesworth, M.C., Goodmanson,Therapy (Eds., Mackiewicz, A. and Sehgal , (P.B. l , M., Zhang, S., Seay, 7. , Klee, G.S.. Tindal l , D.J.

Birkhauser Verlag, Basel, p. 73. and Young, C.Y. (1 9971. Cancer Res., 57 : 2651 .

6. Ader, R., Cohen, N. and Fel ten, D. (19951. 17. Diamandis, E.P. and Yu, H. (1 997). Urol . Cl in.

Lancet. ,345 : 99. North Am., 24 : 275.

7, Ratnakar, N., Sharma, 8.K., Chaturvedi , K.U., 18. Werb, 2. , Ashkenas, J. , MacAuley, A. and

Kaur, S. , Naik, S.L.D. and Ray, A. (19981. Wiesen, J.F. (1996). Braz. J. Med. Biol . Res., 29 :

A.P.M.I .S. ,1O6 : 1075. 1087.

8. Key, T.J.A. and Beral , V. (1992). ln, Mechanisms 19. Chat ikhine , V.A., Chevr ier , A. , Chauzy, C.,

of Carcinogenesis in Bisk ldentif ication (Eds., Duval. C.. d'Anjou, J., Girard, N. and Delpech, B.

Vairr io, H., Magee, P.N., McGregor, D.B. and (1994) ' Cancer Lett ' ,77 i 51 .

McMichael, A.J.l, l.A.R.C. Scientific Publications,

Lyon,p.255. 20' Heiny,8.M., Albrecht, V. and Beuth, J. (19981.

Anticancer i9es.. 18 : 583.

9. Sharma, B.K. and Ray, A. (2O0Ol. lnd. J. Clin.

Biochem., 15(Suppl . ) :110. 21 . van der Pompe, G., Antoni , M.H. and Hei jnen,

C.J. (1996). Psychoneurcendocrinology, 21 : 361 .

10. Feigelson, H.S. and Henderson, B.E. 11999). In

Endocrinology of Breast Cancer (Ed., Manni, A.). 22. Dorgan, J.F., Longcope, C., Stephenson, H'E', Jr.,

Humana Press, New Jersey, p. 55. Falk, R.T., Mi l ler , R., Franz, c. , Kahle, L"

Campbel l , W.S., Tangrea, J.A. and Schatzkin, A.

11. Ray, A., Naik. S.L.D., Kat iyar, S. , Kumar, A. , {1997}. Environ. Heal th Perspect, 105 (suppl ' ) :

Murthy, N.S., Sharma, S., Bahadur, A.K., Pasha, 583.

S.T., Hus,ain, S.A. and Sharma, B.K. (200O1. /nd'

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B'K. (2OOO). lnd. J. Exp. Biol.,38 : 663.

34

lnd. J. Med. Biochem., 2001, 5(1&21

Study of Brain Damage in Rats in Undernutrit ion

C.Y. Dhume and S.P. Chakrabart i 'Department of Biochemistry, Goa Medical college, Bambotim - Goa - 4o32o2

rUniversity College of Medicine, Calcutta - 70OO20

AbstractBrain damage caused by experimental undernutrit ion was studied in albino rats. The results revealedthat on the gth post-natal day there was 79% decrease in body weight of undernourished ratswhich was not modified even after rehabil itation. The brain weight was also significantly lower inundernourished animals. Likewise, total protein, cholesterol and phospholipid contents of the braiJr inundernourished rats were significantly lower. A decrease in protein phosphorylation in thedeveloping brain during undernutrit ion was also found.

35

lntroductionDuring the brain growth spurt period,undernutrition causes a significant reduction inthe brain weight and the body weight ofdeveloping rat pups. There is a signi f icantdecrease in the total proteins, total cholesteroland total phosphol ipids of brain. Proteinphosphorylation is known to be an importantmechanism of cel lular funct ions. In the nervoussystem protein phosphorylat ion has been l inkedto alteration of ion channel activity andmodulat ions of neurotransmit ter release andsynaptic f unctionsr'2. Protein phosphorylationactivated by calcium and phosphatidyl serine(P.S.) is mediated by protein kinase C (P.K.C),

which is abundant ly present in brain, most lyassociated with the particulate fractions3. Sinceprotein phosphorylat ion has been l inked tomodulat ions of several neuronal f unct ionsincluding neurotransmitter release and action ofneurotransmitters through metabotropicreceptorsa'6, investigation of the effect ofundernutr i t ion on a developing rat brain wasthought of. In this study, the brain weight, bodyweight total proteins, total cholesterol, totalphosphol ipids and a systematic extension of theinvest igat ions have been carr ied out ear l ier todetermine the status of protein kinase

C-dependent phosphorylat ion in the developingrat brain during early post-natal undernutr i t ion.

Mater ials and MethodsInbred albino rats kept on laboratory chow andwater were used for this study. The rats wereundernourished by maternal deprivation followingthe method of Culley and MertzT as used byChakrabart i and Shankars. Li t ters consist ing of6-8 pups were selected. Half of the pups from al i t ter were then separated from the dam 14 hoursa day, whereas the rest of the pups were keptconstant ly with the mother throughout. Theschedule of undernutrition was started from day6 after birth, counting the day of birth as day O.The undernutr i t ion was carr ied out up to the 18thpost-natal day. The body weight of the pups

were taken on the 6'h, 1 2th, 18th and 32"d day. Onthe 32"d day (considered as adult) the animalswere ki l led by decapitat ion. The brain t issue wasremoved rapidly from the animal and weighed.The t issue was cut into smal l pieces using ascissor and 9 volumes of homogenizat ion buffer

[containing 32O mM sucrose, 5 mM Hepes andO.1 mM of Phenylmethanesulf onyl f luor ide(P.M.S.F} which was adjusted to pH 7.41 wasadded to i t . The homogenate was poured intoplast ic centr i fuge tubes and centr i fuged at lOOO

Ind. J. Med. Biochem.,2OOl,5(1&21

g for 1O min in a refr igerated REMI C-24centr i f uge using f ixed angle rotor. Thesupernatant was collected and centrifuged at15000 g for 20 min again in the centr i fuge. Thecrude mitochondrial-synaptosomal fraction lP-2Pel let) was thus prepared.

This synaptosomal fract ion (P-2 Pel let l was lysedin 5 mM Hepes lpH 7.41;5 mM EDTA ;1 mMBetamercaptoethanol ( lysis buffer) andmitochondrial-synaptosomal membranes col lectedafter centr i fugat ion at 1,OO,OOO g for 30 min in aBeckman ul tracentr i f uge. The pel lets weresuspended in the lysis buffer (3-5 mg/ml protein)

and used f or protein phosphorylation

experiments.The protein Phosphorylation experiments were

carried out as adapted by Yu. et ale. Thephosphorylat ion of membrane proteins (150-250

micrograms) in presence or absence of Calcium(free conc.O.l mM) and/ or PS (8O

micrograms/ml) was ini t iated by addit ion of 10

microCi. Of Gamma- I32Pl A.T.OP. (3000 Ci/

mmol; mixed with enough unlabel led ATP to give

finaf ATP concentration of 2 micromoles in theincubation mixture) and incubated for 45

seconds. The reaction was then stopped byaddition of electrophoresis sample buffercontaining 3.3o/o SDS and 5o/oBeta-Mercaptoethanol followed by heating in a

boi l ing water bath for 3 minutes. The sampleswere then collected and analysed by

SDS-polyacrylamide gel electrophoresis following

the method of Laemmlito. The gels were stained

in Commassie Bri l l iant Blue fol lowed by

destaining and drying between two wet squares

of cel lophane paper. The dr ied gels were

subjected to autoradiography. All experiments

were performed 4 to 5 times using different

litters. Protein estimation was done by

Lowry-Fol in methodrr. Total Cholesterol was

est imated by' the method of Mc l lwainr2.

Phosphol ipid was measured by convent ional

method. The quantitative assessment of protein

36

phosphorylat ion was done by al igning the dr iedgel with the autoradiogram and cutt ing of theappropriate bands and measuring the radioact iv i tyin each band by scint i l lat ion count ing using atoluene-based scint i l lat ion f lu id.

ResultsThe body weight of the undernourished rats atthe gth post-natal day was approximately 79o/o ofthe control animals. At 18'h day the weight wasapproximately half of the controls. Even afterrehabif i tat ion of 2 weeks, i .e at the 32'd day ofage, the body weight was st i l l s igni f icant ly lowerthan the controls, al though there was somerecovery in the experimental group duringrehabi l i tat ion (Fig 11.The weight of the brain increases rapidly duringthe post-natal per iod. The rate of growth wassignif icant ly higher in the control group between6'h and 12'h day than before and after this period.

A simi lar pattern of increase was seen inundernourished animals. But the absolute weightof the brain and i ts rate of growth was markedlylower in the undernourished animals. In the12-day old pups, the brain weight was about15% fess and those of 18-day old pups was 24o/oless than that of corresponding control pups.

Even on rehabi l i tat ion, there was a stat ist ical lysigni f icant decrease in the brain weight than that

of the control , al though some recovery occurred

during rehabi l i tat ion, (Fig 2).The protein content was increaesd during

development (Fig 3). The rate of increase was

higher in between 6'h and 8'h days than after thisperiod. In the undernourished, the protein content

at 9 'h and 12'h day was lower than that of control

animals and was stat ist ical ly s igni f icant.

However, at 18 and 32 days of age, the protein

content was essent ial ly s imi lar in both the groups

but the total protein content was markedly lowerper brain in the experimental group.

The cholesterol content was also increased

during development (Fig 41. The ' maximum

Ind. J. Med. Biochem., 2001, 5(1&21

increase was found from the 9th to 32nd days. Inthe undernourished group, on the 18th day, thetotal cholesterol content was 9% lower, whichwas statistically significant. But after two weeksof rehabilitation, i.e. on the 32"dday of age, therewas a catch up on the cholesterol content ol thebrain in the experimental group.I t is revealed from the results (Table 1l that thePhospholipid content of the brain was increasedduring development. In the 12 - day oldundernourished pups, there was a decl ine in thetotal phosphol ipid content, which was also seenin the 18-days old pups. When the synaptosomalmembranes from adult rat brain were incubatedwith '32 P [ATP], several proteins werephosphorylated (49,53 and 84 Kda), whereas,one phosphoprotein of 43.5 Kda wasdephosphorylated. Some low molecular weightproteins were also phosphorylated (Fig 5). Mostsigni f icant ly, the phosphorylat ion of the 49 Kdaprotein was impaired in the undernourishedgroup. Some minor phosphoproteins also showeddiminished incorporat ion of 32 P, on rehabi l i tat ionfor 3 weeks, i .e. on 32 day of oge, theimpairment of Protein Kinase C - dependentphosphorylat ion was most ly abol ished (Fig 6).

DiscussionDuring undernutr i t ion the body adapts i tsel f to i tsnutritional insults in such a way that the relativecomposition of the major components of thebrain l ike protein, cholesteroland phosphol ipidsate kept as close to normal as possible tomaintain the normal funct ioning of the brain.However i t is l ikely that there wi l l be subt lemetabol ic and molecular al terat ions that can haveimportant consequences on the structural andfunct ional development of the brain. ProteinKinase C (PKC) - dependent phophorylat ion isimpaired during undernutr i t ion al though theimpairment is apparent ly reversible duringrehabi l i tat ion.Very few reports on protein phosphorylat ion inthe developing brain are avai lable. Therefore i t isdi f f icul t to compare the results. Since protein

37

phosphorylat ion has been impl icated in numerousfunct ions of the brain, the impairment can haveeffects on the structural and functionaldevelopment of brain, such as al teredprol i ferat ion, growth and development ofneuronal processes, development of long termpotent iat ion and neurotransmit ter release.Therefore, a more elaborate study is essent ial onthe status of protein phosphorylat ion in thedeveloping brain during undernutr i t ion.

References1 . Dunkley, P.R. and Robinson, P.J. ' (1 9861. In

'Progress in Brain Reseach' (Gispen W.H. &Routtenberg A. eds. l , Vol. 69, p.273. ElsevierScience Publishers B,V. (Biomedical Divisionl

2. Hugh, C., Hemmings J.R., Nairn A.C., Meguiness,T.L., Huganir R.L. and Greengard, P.11989l,. Fedn.Am. Soc. Exp. Biol. J, 3 : 1583.

3. Narin, A.C., Hemmings, H. C. Jr. and Greengard,P. (19851 ln : Ann Rev. Biochem (Richardson

C.C., Boyer P.D., Dawid LB., Meister A. eds. l ,Vol. 54, Annual Reviews Inc. , Cal i fornia, U.S.A., p

931.4, Wu, W. C.S., Walees, S. 1. . , Nar in, A.C. and

Greengard, P.(1982). Proc. Natl. Acad Sci.,USA79 :5249.

5. Dekker, L.V., De Graan P.E., Spierenburg H., De,

Wit . M., Versteeg, D.H.G. and Gispen, W.H.(1990). Eur. J. Pharmacol., 188 :113.

6. Gray, E.G. and Whit taker V.P. (19621. J. Anat.96

| 79.

Cul ley, W.J. and Mertz, E.T. (19651 Proc. Soc.

Exp. Biol. Med. 118,: 233.

Chakrabarti, S. and Shankar, R. (1 9841 Neurosci.

ler t . , 48 : 109.

Yu. i . Y. , Shin, C.R. and Hanguk Yong Yanghak

Hoej i (19701 Chem. Abstract ,3 : 81.

Laemmfi , U.K. (19801 Nature;277 :680.

Lowry & Folin (1980], Naturet 277 :680 .

Mc. l lwain, P.L. , Eccles, J.C. and Mc. Geer, E.G.(19781 Molecular Neurobiology of the Rat Brain;

Plenum Press, New York.

7.

8.

9.

10.11.12.

Ind. J. Med. Biochem., 2OO1, 5(1&2) 38

Table 1. The phosphol ip id (mg/g t issue) content of the brain of control and undernour ished rat pups.

Days Control Undernourished

6

12

18

32

18.42+O.2417l.

31.331 + 2.5(5)

32.45+5.7171

34.01 + 4.6 (7)

28.3 r 3.19(5)

29.23'r 3.19 (7)

30.95 t 4.O1 (7)

Values given are the tS.D. of the number of observations given in the parenthesis.

ot lo| ' lo2590

Posr uerel Ace (oevs)

Figure 1. Effect of undernutrition on the body weight of adult rats.

o

-9q3ooGt

coNrROr

I

!a

Lr . lI3zsto

9l t t . ! t !a

PosT NATAL aee (oavs;

Figure 2. Effect of brain weight on the brain weight of developing rats

Ind. J. Med. Biochem.,2OO1,5l1&21

Slott202rro

PotT NATAL AGE (DAY9)

Figure 3. Effect of undernutrition on the brain protein content of the developing rats.ta

39

b,)oIF

C'

zId,I,o.

I

grE

Fzll

zCIg

=uFodA

J

Fo

i

I

+

Egr

Qrr

\7I

FzLlFzot

Jod,bJFrtllr,

o

-\,

PO5T NArAL A6E (DAY5)

Figure 4. Effect of undernutrition on the brain cholesterol content of the developing rats.

lnd. J. Med. Biochem.,2OOl,5(1&21

Figure 5. Autoradiographic pattern of synaptosomal membrane phosphoprotein of controlundernour ished adul t rats.

Lane a: Control + Phosphatidyl Serine (80 microgram/ml.) + Calcium2 * (free conc. O.1 mMl.

Lane b: Membrane incubated in absence of Phosphatidyl Serine, but with Calcium2 + (free conc.0.1 mM).Lane c: Membrane incubated in absence ol Calcium2 + but with Phosphatidyl Serine (8O microgram/ml.l.Lane d: Membrane incubated in absence of Phosphatidyl Serine and Calcium2 +.

Lane e: Membrane incubated in presence of Calcium2 * (free conc. O.l mMl and Phosphatidyl Serine

microgram/ml.!.

Lane f: Membrane incubated in absence of Calcium2 + (free conc, O.1 mMl.

Lane g: Membrane incubalbd in absEnce of Calcium2 + and Phosphatidyl Serine,

and

AcknowledgementsThis work is supported by a grant from Roussel Scient i f ic Inst i tute, India. The author wish to thank Prol .

U.M.X. Sangodkar, Dept. of Biotechnology, Goa Universi ty, for al lowing them the faci l i t ies of his

laboratory. The authors further thank Prof. S. Pamnani & Dr. P.P. Nadkarni , Goa Medical Col lege and Dr.

V.J. Monteiro, Goa Medical Col lege for help and suggest ions.

40

180

Ind. J. Med. Biochem.,2001,5(1&21

Comparative Evaluation of Ouality Control MethodsIn an Institute of Eastern Nepal

Dinesh Puri ' and Amit Srivastava, B.P. Koirala Insti tute of Health Science, Dharan, NepatrDept. of Biochemistry, University College of Medical Sciences, Delhi

AbstractEvaluation of procedures used for quality control should be done by clinical chemists to accept or

reject a given method based on its abil ity to meet requirements of the final user. In this study we

analysed the results of a biochemistry laboratory using two different methods of internal quality

control in order to assess their suitabil ity for such laboratories. The laboratory under study is a

moderately equipped one, both work-load wise and resource-wise. lts performance was assessed

using Westgard method of internal quality control and comparision thereof made by the Cusum

technique. The results indicate the suitability of Westgard method for the laboratories having

moderate resources.

41

lntroductionModern medical t reatment increasingly rel ies on

laboratory results, which must be rel iable and

reproducible through standardization of tests

along with regular quality control measures.

Ouality refers to the satisfaction of the needs andexpectations of the users and the customersr.

The users of health care laboratories are doctors

and customers are the ones paying the bills.lmprovement in quality of laboratory performance

obviously has a direct bearing on the quality of

treatment and patient care. With better analyticalquality, a laboratory can eliminate repeat runs

and repeat requests that of ten might be the

cause of wastage of resources. Evident ly,prevent ing such wastage increases the

cost-ef f ectivenesst'3.

A number of methods for quality control have

been described in l i terature. However, most have

been developed in the West and used in

laboratories where use of definitive analytic

methods is common practice. Such methods are

based on a combinat ion of advanced and

sophist icated techniques, such as gas

chromatography mass spectroscopy and isotope

di lut ion technique, and are highly accurate andprecise. However, most hospital setups in thedeveloping countr ies lack such faci l i t ies, andtherefore some of the methods of quality control,that have been reported successful in the West,would not be appropriate for such laboratories.The present study was undertaken to evluateusefulness of two commonly used methods ofinternal quafity control li.e., Westgard multi-rulechart and Cusum technique) in a moderatelyequipped cl inical laboratory of B.P. Koirala

f nst i tute ol Health Sciences, Eastern-Nepal,which could represent the laboratory set up of

most of the hospitals in the developing world.

Mater ials and MethodsThe methods of internal qual i ty control used in

this study, i.e., Westgard multi-rule chart and lhe

Cusum technigue employ a series of control rules

for the interpretat ion of the control data. The

control data f or this study were obtained by

analyzing samples of the control mater ials on 21

days of a month. The control mater ials used in

thc present study were of two kinds ; Pooled

serum : This was obtained by salvaging the extra

lnd. J. Med. Biochem., 2OO1, 5(1&21

serum left over from daily blood samples as per

the WHO guidel inesa Hemolyzed, l ipemic or icter ic

samples, as also the samples showing grossly

abnormal results, were not included; andLyophilized serum : The commercially availableserum obtained from Merck diagnostic of batchnumber HSN 431B was used.Several 1 ml aliquots were prepared from thecontrol materials and stored at sub-zero

temperatures. Each duy, a fresh aliquot was

thawed and analyzed. The parameters estimatedwere glucose, urea, creatinine, total bilirubin,direct bilirubin, uric acid, aspartate transaminase

alanine transaminase, alkaline phosphatase, totalprotein, albumin and calcium. The estimationswere conducted by Merck Vitalab selectra 2

autoanalyzer, except for the total and direct

bilirubin, which were estimated by the manual

method using Ranbaxy diagnostic kit. After

coflecting results tor 28 days, the mean ( ) and

the standard deviatioh (SD) values for theseparameters were calculated, the Levey-Jennings

charts were constructed , and the error was also

determined according to rules of multi-ruleprocedure developed by Westgard and

associates. The control limits were taken to bemean + 1,2 and 3 standard deviations, and theprobability of false rejection was kept very low(0.01 or less) by appl icat ion of the fol lowing

rules :(i) Warning rule :One control observation

exceeding x t 2SD;(ii) Rejection rule : One control observation

exceeding x + 35Dor x-3SD ( l3s), or two

consecutive observations exceeding x+2SD or

x-2SD (26l,or four consecutive observations

exceeding x+1SD or x ' lSD"(4rs), or d i f ference

between two observations within a run > 4SD

(Ror); moreover, ten consecutive values on one

side of the mean also served as rejection rule(1 Ox).Cusum graph was constructed by using the mean

value of each day obtained from the results under

42

considerat ion and the value so obtained wasadded to the previous day's cumulat ive sum. Thisgave the current Cusum value. The Cusum valuesplotted daily will tend to lie on a straight line solong as accuracy is maintained, while any changein accuracy is reflected by change in slope of thel ine.

Results and DiscussionMean values of all the parameters and the controll imits drawn at the mean + 1,2 and 3SD for thepooled serum and HSN-serum are shown in table1. In case of the pooled serum, the measuredvalues of direct bilirubin lay beyond the warningl imit on day 1,3 and 6. The reject ion rule isapplicable on day 22, since ten consecutivevalues l ie on one side of the mean. Urea on day 2and day 22 crossed the warning l imit . Totalprotein on day 3 and total bi l i rubin on day 18also crossed the warning limit, however, in caseof the HSN serum, the same parameters on thecorresponding days were within normal l imits.This implies that these were random errors andnot systematic errors. Simi lar ly, for HSN serum,creatinine on day 4, total protein on day 5 andalbumin on day 24 crossed the warning l imit .This also indicated random errors. Apart fromthese, all other parameters for both types ofcontrol sera were within acceptable range. Inview of the f act that the rules selected areparticularly sensitive to any errors either random

or systematic and owing to less errors detected

in the present study, i t might be concluded that

the results of our laboratory were quite

satisf actory.However, a different picture emerges when theseresufts were analysed by Cusum lcumulativesum) chart, where the statistics plotted was the

algebraic sum of the positive and the negative

deviations of the results from the expected mean

value. The graph was plotted for several bloodchemistr ies (e.g., glucose, utea, alaninetransminase and cholesteroll and is shown in

Ind. J. Med. Biochem., 2001, 511&21

Fig. l . l t was found that the points were notrandomly scattered around the expected value,but rather concentrated on one side so that theCusum value rises (or declines) steadily. Thisindicates erroneous results, more so because atseveral points the Cusum values were too largeto be considered acceptable. Thus, the laboratoryperformance is considered to be unsatisfactoryby this method. lt is interesting to note that thesame results had suggested a satisfactorylaboratory performance with the Westgardmulti-rule procedure. lt seems possible that someminor analytic errors, that were not apparent inthe multi-rule procedure have been identified onapplication of a more advanced technique. For

43

instance, regular upward or downward trends(which indicate that the method is going out ofcontrol) may not be easily apparent on the usualcontrol chart but are usually manifested byplotting accuracy data on a cumulative sumchart.The study suggests the need for taking morecorrect measures for improving laboratoryperformance and also indicates that Cusummethod is too demanding and with moderatelaboratory facilities it may not be possible tomeet the more stringent criteria required by thismethod. Thus, the Westgard techr.rique appearsto be a more suitable method of quality controlfor this type of laboratory having basic facilitiesonly.

Table 1. The mean values of various parameters, the control limits, and the values crossing thesel imits in case of the pooled serum and the commercial ly avai lable lyophi l ized serum (HSN-4318).

Parameters Pooled Serum HSN 4318 Serum

Control Limits Values beyond Control LimitsMean (x) standard control limits Mean (xl SD

deviaion (SD)

Valuesbeyond

control limits

Glucose (mg/dllUrea lmg/dll

Creatinine (mg/dl)

ALT {units/l)AST {units/l}

Total Protein (g/dl)

Albumin (g.dl)

Uric acid (mg/dl)

58.324.2

0.69

16.4136.654.69

3.15

2.65

193.3

161 .6122.3o.76

o.38

10.654.53.

o.21

2.94.581.15

1.OO

o.39

45.3

25.117.8o.17

o.o7

12148.8

1.26

40.9761.66.1 6

4.8

6.43

250

114.1116

2.621.38

NilDay 2 value

114.41<x-2SDDay 22 value

(33.6)>x+2SDlNil

NitNil

Day 3 valuet2.4) < X-2SD

Nil

Day 2 value(1 .8t < X-2SD;Day22 value

13.481>X+2SDNil

Ni lNi l

Day 18 value{1.21>X+2SD

Day1,3,6 values(o.2) < x-2sD

NilNi l

o.11

1.O462.53o.36

o.3

o.33

21 .71

10.337.77

Day 4 value(o.99lx-2sD

NilNi l

Day 5 value{7.41X + 3SDDay 24 value(4.O > X-2SD

Nil

Alkalinephosphatase

(KAU/drlCholesterol (mg/dl lTr iglyceride (mg/dl l

Bi l i rubin (Total l(mg/dl)

Bi l i rubin (Direct)(mg/dl)

Calcium (mg/dl)

Nil

Nitl.til

9.4 1.49 Nil

Ind. J. Med. Biochem., 20O1, 5(1&2)

30

z5

20

15

10

6

nD.tr

{-10

-15.40

-25€0

35

-40

-alt

-50{5

{o{t

-70

-+Glucoso (mg/dtr,

+Urea (mg/dl);

-+ALT (units/L), .

x- Cholesterol (mg/dl)

-75{0

{5

{o

{5

-100

-t05

-110

-116

-120

-1?5

Figure 1. Cumulat ive sum (Cusum) graph of pooled serum for urea, glucose, alanine transaminase (ALT)and cholesterol. Values are shown on Y-axis and days along X-axis.

44

References1. James, O.W. and George, G. Klec 11994) ln:. Tietz

textbook of clinical chemistry, (Eds Earl, A. B. and

Edward, R.A.l, 2nd Edn., W:8. Saunders company,

Pennsylvania p. 548.

2. Me Lauchlan, D.M. and Gowenloela, A.H. 119961 ln:

Varley's Practical Clinical Biochemistry, Eds.

Gowelcola, A.H., Mc Murray, J.R., and Mc Lauchlan,

D.M. 6'h Edn., CBS Publishers and Distributors, New

Delhi , p.290.

3. Kanagasabapathy, A.S., Swaminathan, S. and

Sefavakumar, R (1996) Ind. J. Ctin. Biochem., 11 :17.

4. WHO workshop at Delhi (1997) Small scale

production of quality control sera for clinical chemistry

purposes., Biochem Dept., Langside Glasgow, G12 gTY,

p. l .

5. Boucers, G.N., Bumett , R.W., and Mc Comb(1975) Prep. Materl. Mont. Precis. Clin. Chem.,

21 : 1830.

nil

{

Ind. J. Med. Biochem., 2O01, 5(1&21

Detect ion of Alkaptonuria - A Case Report

Goutam Chakrabort i , Sabyasachi Mull ick, Indranil Chakrabort i ,Santanu Banerjee* and C.R. Maity**Depa^men'i ;H:::Hi'Jl;lli'ril #:1'fi:::llll;,"'o*"'

AbstractA case of alkaptonuria, diagnosed in a male baby of 2 yrs., was reported. He presented with ahistory of black staining of his clothes after being wet with urine, and the black staining wasfurther darkened after washing the clothes with soap. Laboratory tests showed that the patient hada higher level of ur inary homogent is ic acid 1121.1 mg/dl l which is usual ly absent in normal babies.This metabolic disorder, which is rare in Indian population, is due to the deficiency of an enzyme,homogentisic acid oxidase. The patient was discharged with the advice to take 1 gm of ascorbicacid daily.

45

IntroductionAlkaptonuria is a tare, autosomal recessivedisorder result ing f rom a def ic iency of theenzyme homogentis ic acid oxidase (HGA

Oxidaselr . This enzyme catalyzes the oxidat ion ofhomogentis ic acid (HGAI to maleylacetoacet icacid. Over lOOO cases have been described witha high incidence reported in offspr ings f romconsanguineous marr iages and in Czechoslovakia.Only a few reports are avai lable in India2' t . Wereport here a case of this metabol ic disorder in achi ld of 2 yrs. who was invest igated for ur inestaining his clothes black.

Case ReportA 2-year old male chi ld(Fig. 1) presented withhistory of black staining of his clothes after beingwet with ur ine. The stains in the clothes couldnot be removed by washing with soap, rather thestains got darkened. General physical

examinat ion did not reveal any abnormal i ty.There was no cl in ical evidence of anaemia orhepatosplenomegaly. The height and weight werenormal for age. On rout ine examinat ion ur ine was

straw coloured but darkened from surface

downwards on keeping in open air . There was no

bi le sal t , b i le pigment, urobi l inogen, protein orRBC present in ur ine, but Benedicts test or sugar

in ur ine was posi t ive. However, f ur ther tests

showed the absence of sugar in the ur ine.

To evaluate the nature of the pigment, chemical

tests for the presence of melanin, hemoglobin,

myoglobin and indican were done, al l of whichgave negat ive resul ts. When to 5 ml of 3% si lver

ni t rate solut ion was added to O.Sml , ur ine,

fo l lowed by a few drops of 10% ammonia, a

black colour developed. The test was consistent

with alkaptonur ia. The ur ine showed a s igni f icant

concentrat ion of HGA againsta hydroquinone

standard ( in ammonium molybdate and potassium

dihydrogen phosphate buffer) a l 42O nm. Ur inary

HGA concentrat ion was 121.1 mg/dl . On the

basis of these f indings, a diagnosis of

alkaptonur ia was made. The pat ient was advised

ascorbic acid 1 gm/day with plenty of water. He

was also advised to come for a fo l low-up af ter 3

months to monitor his ur ine HGA level .

DiscussionHomogent is ic acid, an intermediate in the

metabol ism of the amino acids, phenylalanine and

tyrosine, is oxidized to maleylacetoacet ic acid by

Ind. J. Med. Biochem.. 2001, 5(1&21

HGA oxidase. The metabol ic defect inalkaptonuria is the complete and congenitalfai lure to synthesize act ive HGA oxidaset. ln thismetabolic defect urinary HGA concentration isincreased due to its rapid secretion in renaltubules without affecting the serumconcentration. Alkaptonuria patients void a urinewhich at f i rst does not show any abnormal i ty incolour. However, the ur ine is alkal ine andcontains a lower amount of ascorbic acid andother reducing substances. The HGA present inur ine is usual ly oxidized in open air to i ts polymer

benzoquinone acet ic acid and the polymerizat ion

is hastened by alkal i . This explains why washingdiapers of alkaptonuric infants with soap make

the stain more intense instead of removing its.Ochronosis is a general ized pigmentat ion

occuring in alkaptonuric pat ients. Usual ly theearliest change that can be detected is thepigmentation of sclera or ear, but these changes

are rarely noticeable before 20 or 30 years ofage. Ochronot ic arthr i t is is a regular

manifestation of long standing alkaptonuria.Hench reported that this arthritis resembles

rheumatoid arthritis clinically but radiologically it

is like that of osteoarthritiss. The earliest

sypmtoms are l imitat ion of motion of hip, knee or

shoulder. The xrays may reveal changes

considered almost pathognomonic ofalkaptonuria. The f i rst paper descr ibing theinheri tance of alkaptonuria was that of Garrod in

1902, in which he presented evidence that this

condit ion is congenital and famil ia l and that i t

occurs more often in famil ies in which there are

consanguineous marraigesT. At present most

46

cases, i f not a l l , appear to represent theinher i tance of a s ingle autosomal recessive gene

located in a 16cM region on chromosome 3028.

Attempts to t reat a lkaptonur ia have been directed

ei ther towards correct ing the under ly ing

metabol ic defects or prevent ing or reversing thepigmentat ion and arthr i t is . In the past i t had been

suggested that dietary phenylalanine and tyrosine

be reduced to decrease the output of HGA. This

is not pract ical and might be dangerous i f

cont inued long. Since from a pract ical standpoint

the importance of th is metabol ic defect is mainly

that i t leads to arthr i t ic changes and

pigmentat ion, the therapeut ic measures would be

aimed pr imari ly at prevent ing these

compl icat ions, Ascorbic acid usual ly protects

lysyl hydroxylase f rom inhibi t ion (v ia

benzoquinoneacet ic acid ? ) and relat ively high

t issue concentrat ion of ascorbic acid might

reduce the degree of pathologic changes in the

connect ive t issues. At least , extended cl in ical

exper ience giv ing a gram of ascorbic acid per day

in div ided doses could be worth invest igat inge.

NTBC[2-( 2-ni t ro-4-tr i f luoromethyl-benzoyl)-

1,3-cyclohexanedionel , inhibi tor of pHppa

oxidase, may also be given as t r ia l ro. The

relat ionship between the metabol ic defect and the

compl icat ions, ochronosis and arthr i t is , remain a

chal lenging research problem of the future. Even

the lack of HGA oxidase would not be the

ul t imate cause of these compl icat ions, and the

mechanisms that br ing them about are st i l l

unknown.

lnd. J. Med. Biochem., 2OO1, 5{1&21 47

Fig.1 Shows the picture of alkaptonuric chi ld.

References

1 . Garrod, A.E. l19O9l lnborn errors of metabolism

Frowde, Hodder & Stoughton, London : Stanbury,

J.B., The metabolic basis of inherited diseases,

McGraw-Hill Book Co., USA, P 308 - 325.

2. Parikh, A., Khubchandani, R.P., et al. (1988) J.

Assoc. Phy. lndia,36 : 565.

Choudhury, H.R., Gokhroo R.K., Arora S.K' et al

(19831 J. Assoc. Phy. lndia,31 : 676.

Varley, H (19881 In : Practical Clinical

Biochemistry, 4'h Edition. CBS Publishers and

Distributors, New Delhi, P 724,

Zanonni, G.V., Lomtevas, M. and Goldfinger, S'

(19691 Biochem. BiophYs. Acta,177 :95,

6. Hench, P.S. (19481 Ann. lntern. Med.28 : 31O.

7, Garrod, A.E. (19O21 Lancet, 2 : 1616.

8. La Du, B.N. et al (19951 Alkaptonuria : Ihe

Metabolic Basis of lnherited Disease lStanbury,

J.B., Wyangaarden, J.8., Fredrickson, D.S. Edl p

308-325.

Woff, J.A., Barshop, B., Nyhan, W.L., Lestic, J.,

et af {19891 Pediatr. Bes. 26: 140.

Scriver, C.R. f2OOll The Hyperphenylalaninemia

and Alkaptonuria : Cecil's Textbook of medicine

(Goldman, L., Bennet, J.C. Ed., Harcourt

Publ ishers, P 111O1.

9.3.

10.4.

5.

I JMBMolecular Epidemiology : New Concept in Biology

The task of epidemiology is to provide a mult i faceted view of a disease, principal ly to guide preventive strategiesand also to formulate targeted treatment options. As a science, i t is inherently mult idiscipl inary. Pathology,microbiology, cl inical medicine as well as social sciences work in tandem in this "bridge" discipl ine to f igure out theet io logy of the disease, i ts 'socio-demographic distr ibut ion, factors in l luencing i ts 'expression and spread in thecommunity and the various potential areas of intervention that may be useful in altering the natural history of thedisease in question. Human diseases form a continuous spectrum - at one end of the spectrum are the classicalgenetic disorders, which shows a simple mendelian pattern of inheritance, althouglr varying in expressivit ies. At theother end of the spectrum are the environmental ly determined diseases l ike infect ious diarrhea, which occurs in thevast majori ty of people exposed to the microbe, which may, however, vary in virulence, In between are the largemajori ty of human ai lments, which are mult i factorial and results from a complex interaction between genetic lactors

and environmental inf luences. Tredit ional ly, epidemiology had focused attention on phenotypic alterat ions in a

disease. A detai led analysis of environmental and phenotypical ly overt genetic inf luences had thus been made based

on the information avai lable from these epidemiological studies.

The last decade had shown tremendous progress in genetics with the discovery of new tools in molecular biology

that have made studies on molecular genet ics s impler, Discovery of Polymerase chain React ion (PGR) in 1986 bi K.

Mull is & co workeis provided the cutt ing edge to the snowball ing science that was al l set to change many

paradigms and prevai l ing concepts in biology. Powered by the molecular revolut ion that i t has ushered in, the gene

had jumped out of the bench to the bedside and the f ields of populat ion epidemiology today. An inadvertent

outcome of these developments in molecular biology is the development of hybrid discipl ines that results from

integration of molecular techniques in tradit ional sciences. Epidemiology has also been caught up in the wind and the

"fusion science' cal led molecular epidemiology has emerged.

Molecular epidemiology has been defined as the science that focuses on the contr ibution of potential genetic and

environmental r isk factors, identi f ied at the molecular level, to the et iology, distr ibution and prevention of disease

within famil ies and across populat ions. The objectives of molecular epidemiology are quite broad and include: (a)

descript ive and analyt ical studies to evaluate host/ environment interaction in disease (b) the development of

prevent ive strategies for the control of bacter ia l , parasi t ic and viral d isorders through molecular diagnosis (c)

prevent iorr 9f norr-communicable diseases and genet ic disorders by assessing r isk and ident i fy ing suscept ib le

indiv iduals.

Because ot i ts ' hybr id nature, the foundat ions of molecular epidemiology are la id by several re lated f ie lds t ight ly

roped together. This includes populat ion genet ics, quant i tat ive genet ics, epidemiology and bio-stat ist ics ' lndeed, the

study of populat ion distr ibution and ethnic variat ion of human genetic (mendelian) diseases is the essence of human

populat ion genet ics. For example, Wi lson's disease has been l inked to mutat ions in a gene on chromosome 14,

named ATP$7 BETA, that encodes a transmembrane protein implicated in copper transport across the golgi

membrane. l t has become clear that not a s ingle isolated mutat ion, rather hundreds of mutat ions al l over the gene

occur in Wi lson's disease, The prevalence of mutat ions also shows ethnic var iat ion; the one prevalent in a caucasian

populat ion is dif ferent from the one most prevalent in an oriental populat ion, In order to develop a diagnostic test i t

is necessary to f ind out th is most f requent mutat ion in a given populat ion and then to t ry to look lor th is in pat ients

samples by molecular techniques. This detai l ing of the f requency and pattern of var ious mutat ions in a given

populat ion to serve as a background informat ion for subsequent studies would be a job of molecular epidemiology.

On the other hand, the vast majori ty of human diseases are not genetic in the classic sense, but are caused by an

abnormal outcome of a complex interaction between the genetic make-up of the host and the environmental factors'

Thus, f inding out the genetically susceptible individuals in such circumstances can lead to preventive interventionsand precautions. Indeed, the avail ibil i ty of the detailed structure and detail ing of the various loci of the humangenome has opened up a golden era for studying the genetic predisposition to such complex multifactorial diseases.To find out the susceptibil i ty gene one needs to look for the several putative gene loci based on preexistingknowledge of phenotypic pathophysiological derangements that occur in the disease in question. The increased riskthat the presence of a specific genetic defect confers on the person to the development of the disease state is ameasure of the strength of the association between the gene and the disease. Such an assessment has to be madein the backdrop of knowledge of overall population prevalence of the loci in question, a principle that so long wasapplied to classical mendelian diseases only. Thus, the relevance of molecular epidemiology in the context ofcommon diseases is expanding day by day. In an ethnically diverse population l ike India, it is expected that many ofthese genetic influences wil l show inter ethnic variabil ity that should be an important area of work for the country.Leaving aside the pivotal role that molecular epidemiology has played in transforming our understanding of commonnon-infectious diseases, the so called genetic epidemiology, the study of infectious pathogens has had a giant leapforward with the availabil ity of molecular tools. Nearly every aspect of our understanding of infectious pathogens isnow understood at the molecular level. This includes diagnosis of infections, particularly when the traditional cultureand staining techniques fail - as do happen most often with viral infections and infections in immuno-suppressedpeople. Molecular diagnosis of infections has opened upon a new area in molecular epidemiology, the mode oftransmission of infections, within a family or a group, can be studied with much more greater precision and claritythan the previous serological methods. Human and microbial organisms have co evolved in the biological world andtracing the segregation of relevant gene flows amongst populations excellently depicts this co evolution in biology.In the case of viruses, RNA viruses particularly, genetic variants often develop at a very high rate and this mightalter the natural history of the disease and modify the response to treatment. lt is important, thergfore, to diagnosethese viral mutants. Molecular typing methods have been developed for microbes and a phylogenetic tree, amolecular pedigree analysis, can be developed that can provide significant insight into evolution of the microbe inquestion. The study of microbial resistance to drugs has also undergone revolution - many of these resistances arecaused by plasmids and other drug resistance genes that can be found out by simple PCR based analysis.Epidemiology sharpened by,the molecular weapon is making strides in science. lt is very l ikely that newer areas ofits' application wil l soon be developed. An important issue in developing countries is the cost involved. A potentiallycomplex issue wil l be application of molecular techniques for simple public health problems in developing countries,because of our inherent tendency to catch hold of any new modality that surfaces. The proper perspective ofapplication needs to be decided in the developing countries ti l l i t becomes much more cost-etfective. lts' t ime thatwe need to develop our human resgurces ready to keep pace with the upcoming revolution.

Dr. Abhi j i t Chowdhury, M.D., D.M.Assistant ProfessorDeptt. of Gastroenterology, IPGMER, Calcutta

References1. Khoury, M.J. , Beaty, T.H., Cohen, B.H. (19931. Fundamentals of genet ic epidemiology. OUP. NY.

2. Smith, C. (19761. Statistical resolution of genetic heterogeneity in familial disease. Ann. Hum. Genet. 39 ;281-290.

3. Risch, N., Merikangas, K. (1996). The future of genet ic studies of complex human disease. Science. 273 :1 51 6-1 5l 7.

4. Thomson, G. {1995). Mapping diseased genes: fami ly based associat ion studies. Am. J. Hum. Genet. ,57 t487-498.

INDIAN JOURNAL OF MEDICAL BIOCHEMISTRY

Editor-in-ChiefProfessor C.R. MaitY

Ex-Officio Members

Prof. C. R. MaitYPresident, A.M.B' l 'Burdwan.

Prof. B. C. KabiNew delhi

Prof. K. Shanti NaiduHyderabad

Prof. S. MazumdarChandigarh

Dr, M. P. Sr idharanCalicut

Prof. B. iM. RudreshaMandya

Prof. V. GovindarajuHony. Secretary, A.M'8.1.Bangalore.

Members of the AdvisorY Board

Prof. S. Chakraborty Prof ' A' Bandyopadhyay

Calcutta Calcutta

Prof. A. S. Saini Prof ' V' Mal l ika

Haryana New Delhi

Prof. R. Raian Prof ' S' ChaudhurY

Cal icut Dhanbad

Editorial BoardDr. S. MahapatraOrissa

Prof. G. P. SinghRohtak

Prof. A. AnandiMadras

Dr. D. DashVaranasi

Prof. H. S. ViruPakshaMysore

Journal AssistantsDr. (Mrs.) A. Roy Dr ' B' Mondal

Calcutta Burdwan

Dr. B. Saha ' - Dr ' B' Bhattacharlee

Durgapur Burdwan

Dr. l . ChakrabortyBurdwan