Transcript
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insightVol. XXIII No. 1 MARCH 2005

Scientific Journal ofMEDICAL & VISION RESEARCH FOUNDATIONS

18, COLLEGE ROAD, CHENNAI - 600 006, INDIA

Editorial

Perspective — Amblyopia Treatment Study: Where do we go from here? — S. Meenakshi

Sodium Potassium ATPase : Sodium Potassium Pump; (Na+ - K+) pump— S. Ramakrishnan

Frosted branch angiitis in renal transplantation: a case report— Sudha K Ganesh, Jyotirmay Biswas, Swapnil M Dongaonkar

Occupational Vision testing – An Introduction — R. Krishna Kumar

Dried Blood Spot (DBS) for Vitamin A analysis – a field friendly method— N. Angayarkanni, A. V. Sai Jyothi, K. Coral, R. Punitham, K. Krishnakumar,Madhava Rao and S. Ramakrishanan

Muscle Puzzle — Anand Vaijwade and S. Meenakshi

Different methods of occlusion

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Amblyopia, yes that age-old problem of the young, has received a lot ofattention recently. The perspective on amblyopia treatment study summarizesand gives the essence. Na K ATPase, an important part of many physiologicalprocesses gets a closer look in the Laboratory sciences article. Vitamin Aanalysis for Vitamin A deficiency is looked into by a novel method, in a pilotstudy. An interesting case report on Frosted Branch angiitis with review ofliterature is presented. Occupational optometry, an essential and field of thefuture is introduced in a lucid fashion. A muscle puzzle that sets you thinkingcompletes the issue.

Dr S MeenakshiEditor

EDITORIAL

SANKARA NETHRALAYA, CHENNAISIR RATAN TATA TRUST COMMUNITY

OPHTHALMOLOGY FELLOWSHIP PROGRAMME

June / August / October / December 2005

CATARACT MICROSURGERY FELLOWSHIP (Basic & Advanced) - 2 months duration.A registered Medical Practitioner with Post Graduate Diploma or Degree in Ophthalmologycan apply.

Bio DataProvide all useful information about yourself in plain paper and send it to theundersigned.

InterviewCandidates called for interview need to appear for MCQ / personal interview at theirown cost. Send application indicating the batch required. For details write to :

Academic Officer, MEDICAL RESEARCH FOUNDATION , 18, College Road,Chennai - 600 006. Fax : 044-28254180 e-mail : [email protected]

Bio Data & Application can be faxed or e-mailed or sent by speed post.

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Introduction:

Amblyopia has traditionally been definedas a decrease in visual acuity caused bypattern vision deprivation or abnormalbinocular interaction for which no causes canbe detected by the physical examination ofthe eye and which in appropriate cases isreversible by therapeutic measures.Amblyopia is said to afflict 1-4% ofpopulation, this number based on largepopulation studies. The rate is even higherin medically underserved populations.

Amblyopia has traditionally been treatedby occlusion and penalization with a fewstudies suggesting medical treatment options.While the effectiveness of both occlusion andpenalization had been proven by severalstudies, individual practice variability in thedose of occlusion, or even the definition ofpart time and full time occlusion promptedthe Pediatric Eye Disease Investigator groupto study amblyopia as one of the areas ofinterest and research.

What is PEDIG?

The Pediatric Eye Disease InvestigatorGroup (PEDIG) is a collaborative networkdedicated to facilitating multicenter clinicalresearch in strabismus, amblyopia and othereye disorders that affect children. Thenetwork, which was formed in 1997, i sfunded by the National Eye Institute (NEI).  The NEI is a part of the National Institutesof Health, which is the branch of governmentthat funds medical research.  There arecurrently over 60 participating sites (offices)with over 120 pediatric ophthalmologists andpediatric optometrists in the United Statesand Canada participating in the network.

The multi center randomized controlledstudies to answer several questions onamblyopia are collectively and popularlyknown as the Amblyopia Treatment Study(ATS).

What is the Amblyopia Treatment Study (ATS) ?

The ATS addressed questions regardingboth moderate and severe amblyopia. Thetherapeutic regimens compared eitheratropine and patching or different amountsof patching. The studies addressed childrenin the 3 to 7 years age group. However aseparate study addressed amblyopia inchildren in 7 to <17 year olds as well.

Like other multicenter trials with visualacuity as an important treatment outcome,the ATS established and published a newprotocol for visual acuity testing during thestudy.

The protocol used isolated surroundedHOTV optotypes (Figure 1). The protocol wasevaluated using the Baylor Video Acuitytester (BVAT). The protocol had a high levelof testability in 3 to 7 year olds and excellenttest-retest reliability.

Perspective:

Amblyopia Treatment Study: Where do wego from here?S. Meenakshi , Sankara Nethralaya ORBIS Pediatric Ophthalmology Learning and Training Centre

Figure 1

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Answers to questions:

The following are several questionsaddressed and answered by the ATS:

1. To compare 6 hours versus full timedaily patching for severe amblyopiadefined as visual acuity in the 20/100 to20/400 range in less than 7 year olds.175 children were studied. The patchingwas combined with one hour of near visualtasks.

Visual acuity in the amblyopic eyeimproved a similar amount in both groups.The improvement in the amblyopic eye acuityfrom baseline to four months averaged 4.8lines in the six-hour group and 4.7 lines inthe full-time group (P=0.45).   The meandifference between groups in log MAR acuityadjusted for baseline acuity = -0.03, 95%confidence interval, -0.11 to 0.05.

The study concluded that for severeamblyopia, prescribing six hours of dailypatching produces an improvement in visualacuity that is of similar magnitude to theimprovement produced by prescribing full-time daily patching in children 3 to less than7 years of age.

2. To compare moderate amblyopes withvisual acuity in the 20/40 to 20/80range, also in the less than 7 years agegroup who receive 2 hours versus 6hours, both treatments combined with onehour of near visual tasks. 189 childrenwere enrolled of who 181 completed 4months follow up. Visual acuity in theamblyopic eye improved a similar amountin both groups. The improvement in theamblyopic eye acuity from baseline to 4months averaged 2.40 lines in each group(P=0.98).  The mean difference in log MARacuity between groups = 0.001, 95%confidence interval, -0.040 to 0.042.

The study concluded that for moderateamblyopia in the studied age group two hoursof patching was as effective as six hours.

3. To compare atropine and patchingtreatments for moderate amblyopia withvisual acuity in the amblyopic eye < 20/40 to > 20/100, intereye difference > 3log MAR lines. The patients were requiredto have worn their refractive correction for4 weeks prior to enrollment. Four hundredand nineteen patients were enrolled.

The initial number of patching hourswas prescribed based on an investigator’susual practice with minimum of 6 hours perday prescribed. After 5 weeks of treatmentpatients prescribed 10 or more hours ofpatching per day had greater improvementin visual acuity compared with atropine orpatients with 6 to 8 hours of treatment.

At six months, visual acuity wasimproved from baseline by about 3 lines ofvision in both the atropine and patchinggroups.  Improvement initially was faster inthe patching group, but after six months,the difference in acuity between treatmentgroups was small.  The mean visual acuity(Snellen approximation) at six months was20/32 in the patching group and 20/32 -2 inthe atropine group.  This small differencebetween groups was considered clinicallyinconsequential. 

Both treatments were well tolerated,although atropine had a slightly higher degreeof acceptability on a parental questionnaire. More patients in the atropine group than inthe patching group had reduced acuity inthe sound eye at six months but this did notpersist with further follow up.

The conclusion is that both atropine andpatching are effective treatments for moderateamblyopia in children in the age range of 3to less than 7 years old.  Patching has thepotential advantage of a more rapidimprovement in visual acuity and possibly aslightly better acuity outcome, whereasatropine has the potential advantage of easieradministration and lower cost. Thereappeared to be a transient decrease in visionin the sound eye in the atropine treatedpatients but this was found to resolve.

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4. The next question answered was onamblyopia recurrence after cessation oftreatment. The study enrolled 156children with successfully treatedanisometropic or strabismic amblyopiawho were younger than 8 years of age andwho had received continuous amblyopiatreatment for previous 3 months and whohad improved atleast 3 log MAR levelsduring the period of continuous treatment.

The study found that approximately onefourth of successfully treated amblyopicchildren experience a recurrence within thefirst year off treatment. For patients treatedwith six or more hours of daily patching aweaning to two hours prior to cessationreduced recurrence.

5. Evaluation of amblyopia treatment in the7 to <17 year olds was the next issue.The aims were to study the effectivenessof amblyopia therapy in the olderamblyopes and the rate of recurrence aftercessation of therapy. The study concludedthat for patients 7 to <13 years old,prescribing 2 to 6 hours per day ofpatching with near activities and atropinecan improve visual acuity even if theamblyopia has been previously treated. For patients 13 to <18 years old,prescribing patching 2 to 6 hours per daywith near activities may improve visualacuity when amblyopia has not beenpreviously treated but appears to be oflittle benefit if amblyopia was previouslytreated with patching.  

The studies also found that atropine waspsychologically better tolerated.

Unanswered questions:

There are however several unansweredquestions concerning amblyopia, which theATS did not clearly address or have indicatedthat they need further study. Some of theirsand some of ours are as follows:

1. The nature of near activity proposed aspart of the treatment regimen is unclear.The impact of the same on the outcome is

also unclear. This was also not studied bythe ATS.

2. The regimen for tapering of occlusion isan area in need of further study.

3. Weaning off atropine to prevent recurrenceof amblyopia is an area that needs furtherlooking into.

4. Will there be ethnic variation in theresponse to treatment? Can these resultsbe generalized to other countries?

5. Is atropine safe in tropical countries withmore sunshine throughout the day wherechildren may not be able to affordprotective sunglasses?

6.Is there a longer period of refractiveadaptation in many children, which needto be studied separately before institutingany amblyopia therapy?

7.In other causes of amblyopia such asdeprivational, can these results beextrapolated?

Further studies are needed both by thePEDIG and other similar groups of pediatricophthalmologists in other parts of the world.

References:

1. Pediatric Eye Disease InvestigatorGroup. A randomized trial of patchingregimens for treatment of severeamblyopia in children. Ophthalmology2003; 110:2075-87.

2. Pediatric Eye Disease InvestigatorGroup. A randomized trial of patchingregimens for treatment of moderateamblyopia in children. Arch Ophthalmol2003; 121: 603-11.

3. Pediatric Eye Disease Investigator Group.The clinical profile of moderate amblyopiain children younger than 7 years. ArchOphthalmol 2002; 120: 281-287.

4. Pediatric Eye Disease InvestigatorGroup. A randomized trial of atropine vspatching for treatment of moderateamblyopia in children. Arch Ophthalmol2002; 120: 268-278.

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5. Pediatric Eye Disease Investigator Group.A comparison of atropine and patchingtreatments for moderate amblyopia bypatient age, cause of amblyopia, depthof amblyopia, and other factors.Ophthalmology 2003; 110:1632-1638.

6. Pediatric Eye Disease Investigator Group.The course of moderate amblyopia treatedwith patching in children: experience ofthe Amblyopia Treatment Study. Am J Ophthalmol 2003; 136:620-629.

7. Pediatric Eye Disease Investigator Group.The course of moderate amblyopia treatedwith atropine in children: experience ofthe Amblyopia Treatment Study. Am J Ophthalmol 2003; 136:630-639.

8. Pediatric Eye Disease InvestigatorGroup. Impact of patching and atropineon the child and family in the amblyopiatreatment study.  Arch Ophthalmol 2003;121:1625-1632.

9. Pediatric Eye Disease Investigator Group.Two-year follow up of a six-monthrandomized trial of atropine versuspatching for treatment of moderateamblyopia in children. Arch Ophthalmol2005; 123:149-157.

10. Pediatric Eye Disease InvestigatorGroup. The clinical profile of moderateamblyopia in children younger than 7years. Arch Ophthalmol 2002; 120: 281-287.

11. Pediatric Eye Disease InvestigatorGroup. A randomized trial of atropine vspatching for treatment of moderateamblyopia in children. Arch Ophthalmol2002; 120: 268-278.

12. Pediatric Eye Disease Investigator Group.A comparison of atropine and patchingtreatments for moderate amblyopia bypatient age, cause of amblyopia, depthof amblyopia, and other factors.Ophthalmology 2003; 110:1632-1638.

13. Pediatric Eye Disease Investigator Group.The course of moderate amblyopia treatedwith patching in children: experience ofthe Amblyopia Treatment Study. Am J Ophthalmol 2003; 136:620-629.

14. Pediatric Eye Disease Investigator Group.The course of moderate amblyopia treatedwith atropine in children: experience ofthe Amblyopia Treatment Study. Am J Ophthalmol 2003; 136:630-639.

15. Pediatric Eye Disease InvestigatorGroup. Impact of patching and atropineon the child and family in the amblyopiatreatment study.  Arch Ophthalmol 2003;121:1625-1632.

16. Pediatric Eye Disease Investigator Group.Two-year follow up of a six-monthrandomized trial of atropine versuspatching for treatment of moderateamblyopia in children. Arch Ophthalmol2005; 123:149-157.

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It was in 1957, Jens Skou found thatplasma membranes of higher eukaryotescontained the enzyme sodium potassiumATPase (Na+ - K+ ) - ATPase. It is a protein ofmolecular weight 330 x kD with two α andtwo β sub units as ( aβ)2 tetramer. The twolarge α subunits are identical, each with amolecular weight of 110 @ kD and notglycated. They traverse the plasma membranewith eight transmembrane α helical segmentsand two large cytoplasmic domains. The αsubunits have the catalytic activity and ion-binding sites. The two β subunits are alsoidentical, each with a molecular weight of55∆ kDa. Each β subunit has a singletransmembrane helix and a large extracellulardomain with carbohydrate moieties on theirextra cellular domain. The function of βsubunits is not known.

Cross links are possible between α andα or α and β but not between β and β,suggesting that the α units are close to oneanother while the β units are far apart (figure1).

Each large α subunit contains ATPbinding site for the hydrolysis of ATP and asite each for the binding of cardiotonicsteroid inhibitors like digitogenin andouabain. ATP binding site is on the cytosolside of the membrane while the steroid -inhibitor site is on the extracellular space.

The enzyme complex as a whole has onebinding site for phosphorylation, three sitesfor Na+ located possibly at the interfaces ofthe α subunits and one binding site forsteroid inhibitor.

x 270 kD@ 95 kD Earlier report∆ 40 kD

The enzyme has sulphydryl groups,which could undergo oxidation to disulphidebridges even under physiological conditions.Glutathione brings it back to its thiol-containing structure.

2 Enz-SH Enz-S-S-Enz

2GSH

Function of (Na+ - K+) ATPase; the (Na+ - K+)Pump:

The enzyme is omnipresent in the bodyin biomembranes and is a component of aspecific transport system called sodium -potassium pump (Na + - K + ) pump, whichhelps in the maintenance of ionic gradientsof Na+ - K+ occurring between the inside andthe outside of the cells. While the inside ofthe cells has a concentration of K + as highas 140 mM, the extra cellular fluid (ECF)has only 4 mM. Thus K + is mostlyintracellular. On the other hand, intracellular

SODIUM POTASSIUM ATPaseSodium Potassium Pump; (Na+ - K+) pump

S. Ramakrishnan , Prof. & Head, Biochemistry Research Department, Vision Research Foundation,Sankara Nethralaya, Chennai - 600 006.

Figure 1:Na + and K+ ATPase - position andcomposition (Courtesy: Voet et al

Fundamentals of Biochemistry)

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Na+ is only 12mM while its concentration inECF or plasma is 145 mM.

While earlier literature was givingseparate identities of (Na+ - K+) ATPase and(Na+ - K+) pump describing the enzyme as acomponent of the pump-both of them beingoriented in the same membrane, presentlythe enzyme itself is often called (Na + - K+ )pump because it pumps Na+ out and K+ intothe cell getting the energy required with aconcomitant hydrolysis of intracellular ATP.

The overall stochiometry of the reaction is3Na + 2K + + ATP + H 2O → 3Na+ + 2K +

(inside) (outside) (outside) (inside)

Such an antiport of Na + and K+ causescharge separation across the biomembranesince three + (positive) charges exit the cellfor two + (positive) charges entering the cell.(figure 2)

Figure 2:Differential distribution of Na + and K +

in and out of cell (Courtesy: Lehninger'sPrinciples of Biochemistry)

Because of the difference in theconcentration of the same ions inside andoutside maintained by the pump, a diffusionpotential is set up producing bioelectricity tothe tune of about -50 to -70 mV usuallyapproximated to -70 mV. Thus, the shifts ofions due to pump is electrogenic (i) it causesa voltage difference across the membrane and(ii) negative charges on the inside of the

membrane. A voltage difference of - 70 mVcorresponds to the diffusion potential of K +

electrode with 140 mM inside and 4 mMoutside. It should not be forgotten that Na +

also could contribute to bioelectricity as Na +

also has diffusion potential brought aboutby different concentrations inside andoutside.

(i) Bioelectricity brought about by (Na+ - K+ )ATPase is of paramount importancewithout which no life could exist. Thebiochemical, biophysical and physiologicalevents in living systems are governed to aremarkable extent through the force ofbioelectricity. If the nature or magnitudeor both of this electricity were affected,pathological states would follow.

(ii)The extrusion of Na+ enables animal cellsto maintain their cell volume and controltheir water osmotically. In the absence ofthe enzyme (pump) or non functioning ofthe pump to maintain a low internal Na +

ion concentration, water would osmoticallyrush in, to such an extent that animalcells which lack cell walls would swell andburst.

In the eyes, due to intra lenticularaccumulation of Na + with non functioningpump, water will be imbibed. As a result,the extent of hydration will increase. Thismay end up with cataract (figure 3), that is

Figure 3:Sodium pump to ward off cataract

(Courtesy: Ramakrishnan's OcularBiochemistry)

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why there is (Na + - K+) pump only in theepithelium of the lens which section alonepossesses mitochondria and producessignificant amounts of ATP needed for theoperation of the pump. Rest of the lens hasno mitochondria and there is no TCA cycleexcept in lens epithelial cells.

Likewise corneal deturgescencenecessary for the passage of light throughits stroma will be disturbed by theaccumulation of Na+ followed by water in thestroma. This is prevented by the cornealendothelial (Na+ - K+) ATPase. Infact, thereare five endothelial pumps including (Na + -K+) pump in the cornea. Sodium and throughit, water are evicted and corneal transparencyis maintained.

During action potential of cells, there isan influx of Na + into the cells and therbyreversal of polarization (usually expressed inphysiology as depolarization). There willensue reversal of electrical changes.Convulsions are a result of a large influx ofNa+. Acetyl choline, Glutamate, etc., whichare neuro transmitters facilitate the entry ofNa+ in the neuronal cells and help in thetransmission of nerve impulses along theaxon through a wave of electronegativity. Butafter the action proper physiological orpathological, it is the (Na+ - K+) pump whichsets right the disturbance in the ionicdistribution and rehabilitates the cells to theirnormal status.

Just as in the brain, the excitability ofthe muscle cells also disturbs the ionicdistribution. These cells also owe to (Na + -K+) pump for their revival to normalcy.

The resting cells are reported to spend25 to 33% of ATP synthesized by them forthe operation of (Na+ - K+) pump. Nerve cellsgo to the extent of spending 70% of ATP forthe purpose.

A doubt may arise on the necessity ofoperation of the pump under normalcircumstances. It is necessary because, dueto the concentration gradient, Na+ ions fromhigher concentration in ECF would diffusepassively into the cells with a lower

concentration of Na+. This would increase theintracellular Na+ which has to be corrected.This is done by the (Na + - K+) pump.

In the Rod outer segment (ROS) of theretina, there are channels for Na +. Only ifthe channels are open and Na + enters theROS, there will be depolarization and neuronswill sense the visual message and transmitto the brain. Cyclic GMP binds to thechannels and opens them up. So they arecalled “gated” channels (figure 4). Thus, thereare many situations in the body when Na +

continuously enters the cells and threatensthe normal distribution of ions. It is in suchcases Na + - K+ ATPase has to come to therescue.

Figure 4: cGMP-gated Na + channel(Courtesy: Berman's Biochemistry of the eye)

MECHANISM OF ACTION OF (Na+ - K+) ATPase

In understanding the mechanism ofaction of (Na+ - K+) ATP ase, the followingquestions come to our mind.

1. How does the enzyme carry Na + ionsoutwards from inside the cells ?

2. How does the enzyme release Na + ions inECF ?

3. How does the same enzyme carry K+ ionsinwards into the cells?

4. How does it release the K+ ions inside thecells?

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K+

Na+ = 0 mm AT

Pase

act

ivity

Na+ = 20 mm

The sequence of events are as follows.

1. Transporter in the enzyme in its E1 stateof conformation binds three Na+ ions insidethe cell and then binds ATP to yield anE1.ATP.3 Na + complex.

2. ATP hydrolysis produces ADP with a high-energy aspartyl phosphate intermediate.

E1 ~ P. 3Na+

Enzyme Protein

— H.N.– CH – CO —

|CH2

|

CO

O|

0 = P – O¯

| O¯

3. This high energy intermediate relaxes toits low energy conformation of the enzyme(E2) forming E2 ~ P. 3Na+ and releases thethree bound Na+ ions outside the cell.

4. E2 ~ P binds to K+ ions from outside thecell to form E 2 ~ P. 2K+ complex.

5. How is the enzyme assisted by ATP indonating the energy required for the activetransport of ions wherever it is againstconcentration gradient?

The answers to the above questions are:

1. Conformational changes of the enzyme (E)to E1 and E2.

2. Orientation of high affinity sites of thelarge sub unit α o f E1 in the intra cellulardomain i.e. high affinity sites face insideof the cell.

3. Orientation of high affinity sites of thelarge sub unit α o f E2 in the extra cellularportion i.e. high affinity sites face theoutside of the cell.

4. Phosphorylation at ATP – binding site afterbinding of 3 Na+ and in the presence ofMg 2+ .

Dephosphorylation by hydrolysis afterbinding of K+ .

E1 is stabilized by phosphorylation.

E2 is stabilized by dephosphorylation

Na+ triggers phosphorylation

K+ triggers dephosphorylation

E1 to E2 and E2 to E1 are by eversion.Ion binding cavity in E1 faces inside the cell.Ion binding cavity in E2 faces outside thecell. Before release of bound Na+ and that ofbound K+, there are conformational changesof enzyme i.e. E 1 to E2 and E2 back to E 1which are brought about by eversion.Conformational differences need not be large.Shift of a few atoms by a distance of as lowas 2Aº might suffice to alter relative affinityof the cavity for Na+ and K+ and change itsorientation. Covalent modification throughphosphorylation can readily produce changesof this magnitude.

It has to be remembered that in thesequence of events described below,hydrolysis of ATP takes place only if Na+ andK+ are present.

β aspartylphosphate

~

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5. The phosphate is hydrolysed to yieldE2.2K +.

6. E2.2K + changes its conformation to E 1back and releases its two K + ions insidethe cell.

E1 can now take up three Na + ions tocontinue the transport cycle. (Figures 5, 6,7)

Figure 5:Na + - K + pump - sequence of events

E1 (of E) E1 • ATP • 3 Na+ E1 ~ P.3 Na +

3 Na+ ~ (P)

~ (P)

Conformational change, eversion to low energy E2

Release of 3 Na+ to outside

E2 ~ P • 3 Na+ E2 ~ P 3 Na+

(outside)

3 Na+ ATP

3 Na+ (P) ~

3Na+ (inside)

then ATP

Inside Outside

Phosphorylation

ADP

Inside Outside Inside Outside

2 K+ ~ (P)

2 K + Binding of 2K+ outside Hydrolysis

E2 ~ P • 2 K+ E 2 • 2 K+

(P)

Inside Outside

2 K+

Conformational change, eversion to E 1

Release of 2 K +

E1 • 2 K+ E1 ready for accepting 3 Na+

2 K + (inside)

Inside Outside

To continue the cycle

Figure 6: Schematic DiagramConformations and functions of

Na + - K + ATPase

E1

E1 • ATP • 3Na+

E1 ~ P •3Na+ Conformational change E2 ~ (P) 3Na+

3 Na+ to outside

E2 ~ (P)

2K+ from outside E2 ~ (P) • 2K+ Hydrolysis of (P)

E2 • 2K+ Conformational change E1 • 2K+ E1

Inside Outside

Inside Outside

ADP

(P)

(P)

(P)

(P)

(P)

2K+ to inside

°°°

°°°

°°°

°°°

� �

� �

� �

� �

°°° 3Na+, then ATP

Figure 7:Conformations and functions of

Na + - K + ATPase

Though each of the reaction steps isindividually reversible, the cycle circulatesonly in the clockwise direction under normalphysiological conditions. This is because, ATPhydrolysis and ion transport are coupledvectorial (unidirectional) processes. Thevectorial nature of the reaction cycle resultsfrom the alteration of some of the steps ofexergonic ATP hydrolysis with some of thesteps of the endergonic ion transport process.

Maximal turn-over number of ATPasemolecules is about 100 sec- 1

Inhibition of (Na+ - K+) ATPaseCardiotonic steroids are specific

inhibitors of (Na+ - K+ ). ATPase and thereby(Na+ - K+ ) pump. Digitogenin and ouabain

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Digitogenin Ouabain

C

B

H HO

3

17 14

OH

H O | ||

C C | CH2 O

C

C

O || C C | CH2 O

H |

OH

OH Sugar O

CH2OH OH

contractility of the cardiac muscle.

It is interesting to note that even beforethe enzyme was found out, William Witheringused Foxglove plant extract to treat congestiveheart failure as early as 1785.

Ouabain which was once thought to beproduced only by plants has recently beendiscovered to be an animal hormone that issecreted by the adrenal cortex and functionsto regulate cellular Na+ and overall body saltand water balance.

The enzyme is also inhibited by nmlevels of vanadate V 5+ ions. Pentavalent ionlocks the E 2 of E. Vanadate structure isanalogous to the biphyramidal structure ofphosphoryl group.

Structure of V 5+

Biphiamidal array of ligands aroundcentral vanadium ion is like that aroundPhosphorus atom during hydrolysis ofphosphoryl group.

are called cardiotonic steroids as they haveprofound effect on the heart. There isstructural similarity of the two in that bothof them have 5 or 6 membered unsaturatedlactone ring with b configuration at C-17, ahydroxyl group in C-4 and C is fusion ofrings C and D. Ouabain has sugar resides atC 3 but these do not contribute to inhibition.

The reaction of inhibition is thehydrolysis of E ~ P

K+ steroidE2~P + H2O E1 + Pi

i.e. inhibition at the level ofdephosphorylation. An important conditionis that the steroids should be available atthe outside face of the membrane to enablethem to bind to the specific sites in the Asubunits of the enzyme.

Digitalis, the extract of the leaves ofFoxglove plant contains digitogenin andouabain.

These steroids increase the force ofcontraction of heart muscle and are thechoice drugs in the treatment of congestiveheart failure. Inhibition of (Na + - K+ ) pumpleads to a higher levels of Na+ inside the cellwhich, in its turn, increases the level ofintracellular calcium. Such an increase ofintracellular calcium enhances the

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Reference:

1. Berman E.R. Biochemistry of the Eye.Plenum Press, New york and Londen,1991; 420.

2. Lehninger principles of Biochemistry, 3rd

edn Nelson DL and Cox, M.M. MacmillanWorth. 2002;420-421.

3. Murray RK, Granner DK, Mayes PA ,Rodwell VM. Harper’ Biochemistry,Appleton and Lange 1991,458.

4. Ramakrishnan S. Essentials ofBiochemistry and Ocular Biochemistry,1992;29.

5. Ramakrishnan S, Prasannan KG, RajanR. Text book of Medical Biochemistry 3rd

ed. Orient Longman, Chennai. 2001;649.

6. Stryer L, Biochemistry, W.H. Freeman andCo. New York. 1981,865.

7. Voet D, Voet JG, Pratt CW, Fundamentalsof Biochemistry. John Wiley and sons Inc.Newyork. 1999;271-273.

AN APPEALA lot of things in this world depend on money - security, shelter, education and evenhealth. But at Nethralaya, money has ceased to be a pre-requisite for sight.

Day after day, year after year, Nethralaya treats hundreds of patients absolutely freeof cost and gives them back their sight.

Yet there is no discrimination between the free patient and the one who pays. Apartfrom the treatment, food, medicines and travel expenses are absolutely free.

Those free patients depend on Nethralaya, and Nethralaya depends on you.

So, come and join the SANKARA NETHRALAYA OM TRUST INC.

For questions about tax exempt status and contributions, please contact:Mr S V Acharya, Treasurer

Sankara Nethralaya OM Trust Inc.14613, Pommel Drive, Rockville, MD 20850, U.S.A.

Phone: (301)251 0378INTERNET e-mail : [email protected], [email protected]

www.omtrust.orgFor those of you in India and elsewhere, please contact:

Dr S S Badrinath, President & ChairmanSankara Nethralaya

(UNIT OF MEDICAL RESEARCH FOUNDATION)18 College Road, Chennai 600 006

Phone: 2826 1265, 2827 1616 Fax: (044) 2825 4180, 2821 0117INTERNET e-mail : [email protected]

LOOK US UP ON THE WEB at http://www.sankaranethralaya.org, http://www.omtrust.orgGIVE ONLINE @ www.icicicommunities.org

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On presentation his BCVA was 6/9 N6in both eyes. Slit lamp examination of righteye revealed a mild nongranulomatousanterior uveitis, aqueous flare 2+ aqueouscells 2+, and vitreous cells in the retrolentalspace. Slit lamp examination of left eye wasnormal. The intraocular pressure byapplanation tonometry was 14 mm of Hg inboth eyes.

Fundus examination with indirectophthalmoscopy of the right eye revealedfrosted branch angiitis along thesuperotemporal vessels and features of CMV

Frosted branch angiitis in renal transplantation:a case reportSudha K Ganesh, Jyotirmay Biswas and Swapnil M Dongaonkar

Ito and co-workers first reported a raretype of retinal vasculitis in a 6-year-old boyfrom Japan.1 Though the initial reports werein the younger age groups around 9 yrs, thedisease has also been reported in older agegroups between 20-29 years in otherwisehealthy individuals. These patients have beenfound to have acute bilateral or unilateralvisual disturbance with signs of anterior andposterior segment inflammation. The fundusappearance is dramatic with swelling of retinaand severe sheathing of the retinal vesselscreating an appearance of frosted branchesof a tree. There have been reports of frostedbranch angiitis in Rubella4 and in AIDS. 5,6

We report a case of FBA and CMV retinitisfollowing immunosupression for renaltransplant Uveitis seen at a referral Uveitisclinic at Sankara Nethralaya Chennai, India.

CMV retinitis is the most commonopportunistic infection in immune deficiencystates, that occurs due to AIDS or due to apharmacological immunosuppression tosupport organ transplantation or to treatmalignancies.

CMV retinitis is the most common ocularopportunistic infection in organ transplantpatients. CMV retinitis occurs in 1-5% ofrenal transplant recipients. 1

CASE REPORTA 29 year old, male reported to our

institute with complaints of floaters anddefective vision in the right eye for the past6 weeks. The left eye was asymptomatic. Thepast medical history revealed that he hadundergone a renal transplant 6 months back.He was on immunosuppressive medication,with Cyclosporine 400mg per day andAzathioprine 150mg per day and oral steroids30mg per day for the past 6 months tosupport the renal transplant.

Figure 1 – Right eye showing CMV retinitisand frosted branch angiitis

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retinitis in the inferotemporal quadrant (Fig.1). Fundus of left eye showed early featuresof FBA along the inferotemporal arcade. (Fig. 2)

Preliminary investigations revealed thatthe leukocyte count was 4,400 cells/cmm,the differential count was normal, and theliver function tests and renal function testswere within normal limits. The ELISA for HIVwas negative, ELISA for CMV IgM was +ve,and CD4cell counts were 363 cells per cmm.

Patient was started on IV Ganciclovir 5mg/kg body weight for 3 weeks andsimultaneously his immunosuppressives weregradually tapered to 200mg of cyclosporineand 75mg of azathioprine per day under thesupervision of a nephrologist.

He was reviewed on 10 th day, at end of3 weeks, and at end of 6 weeks. His visionimproved to 6/6 N6 in both eyes withdramatic resolution of both anterior andposterior segment inflammation (Fig.3). HisCD4 counts on last review were 883 cells/cmm. Subsequently his immunosuppressivetherapy was monitored with CD4 counts, andperiodic ophthalmic reviews.

Discussion:Frosted branch angiitis (FBA) is an

uncommon syndrome previously described asa primary immunologic response inimmunocompetent patients that respondedwell to systemic steroid therapy. 1,3

Subsequently FBA was reported as asecondary phenomenon in immuno-compromised AIDS patients in associationwith CMV retinitis where it was thought tobe a direct infection of the retinal vesselsand the adjacent retina by the CMV virus.This type of FBA responded to anti CMVtherapy3 , 4.

CMV retinitis is the most commonopportunistic infection in immune deficiencystates, which may be due to AIDS or due toa pharmacological immunosuppression tosupport organ transplantation or to treatmalignancies.

CMV retinitis is the most common ocularopportunistic infection in organ transplantpatients. CMV retinitis occurs in 1-5% ofrenal transplant recipients. 1

There are two studies on CMV retinitisin non- HIV patients with chemotherapeuticimmunosupression and all these patientshave responded to anti CMV therapy. 6,7

Wagle7 et al have reported frosted branchangiitis with CMV retinitis in 6 out of the 15patients with CMV retinitis following organ

Figure 2 – Left eye showing frosted branchangiitis

Figure 3 – Montage showing resolution inthe right eye

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organ transplant patients but also periodicallyscreen the eye for evidence of CMV retinitisor FBA. In our case that had CMV retinitisand FBA following a renal transplantation andimmunosupression, w hen the immuno-suppressives were tapered, withoutjeopardizing the transplant, it resulted inimproving the immune status and increasedthe CD4 counts and this in conjunction withganciclovir therapy hastened the resolutionof CMV retinitis or FBA.

References:1. Ito Y, Nakano M Kyu N, Takeuchi M.

Frosted branch angiitis in a child. Jpn JOphthalmol 1976; 30:797-803.

2. Porter R, Crombie AL, Gardner PS, UldallRP. Incidence of Ocular Complications inPatients Undergoing Renal Transplantation.Br Med J 1972; 3:133-136.

3. Kleiner R, Kaplan HJ, Shakin JL,Yannuzzi LA, Crosswell HH, McLean WC.Acute frosted retinal periphlebitis. Am JOphthalmol 1988;106:27.

4. Biswas J, Fogla R, Madhavan HN.Bilateral frosted branch angitis in an 8year old Indian girl. Retina 1996;16:444-45.

5. Spaide RF, Vitale AT, Toth IR, Oliver JM.Frosted branch angitis associated withcytomegalovirus retinitis. Am JOphthalmol 1992;113:522-28.

6. Kuppermann BD, Petty JG, Richman DD,Matthews C, Fullerston SC, Rickman LEet al. Correlation between CD4+ countsand prevalence of cytomegalovirusretinitis and human immunodeficiencyvirus-related non-infectious retinalvasculopathy in patients with acquiredimmunodeficiency syndrome. Am JOphthalmol 1993;575-82.

7. Wagle AM, Biswas J, Gopal L, MadhavanHN. Clinical profile & immunological statusof CMV retinitis in organ transplantpatients. Ind J Ophthalmol 2002; 50: 115-121.

8. Daikos G, Pulido J, Kathpalia SB, JacksonGG. Intravenous & intraocular ganciclovirfor patients with AIDS or chemot-herapeutic immunosuppresion. Br JOphthalmol 1988; 72: 521-524.

transplant in their study. However our patienthad features of frosted branch angiitis andCMV retinitis in the right eye and features offrosted branch angiitis in the left eye.

Our patient was pharmacologicallyimmunosuppressed to support a renaltransplantation. He presented with thesecondary form of FBA and required anti CMVtherapy. We also tapered his immuno-suppressive medications in order to improvehis immune status. This resulted in rapidresolution of CMV retinitis, which hadpresented as FBA.

In patients with AIDS the irreversibilityof the immunosupressed status allows theCMV retinitis to progress. However with theadvent of HAART therapy that improves theimmune status of the patient CMV retinitisis becoming a rarity but is still at large inthe developing countries where HAARTtherapy is unaffordable.

Most studies accept that the CMVretinitis in organ transplant is due to directspread from other patients or transfusion ofdonated blood containing CMV virus.2. Porteret al in their study of 39 patients of allogenicrenal transplant found 2 patients (5%)developed CMV retinitis and 35 patients(87%) had detectable complement fixingantibodies to CMV.

Since the CMV virus is present in thesera of patients having renal transplant,patients at risk are those who have had highdoses of immunosuppressives for prolongedperiods of time.

The systemic risk factor associated withCMV retinitis especially in AIDS is a low CD4count less than 50 cells per microlitre. Inour patient the CD4 count was more than300 cells per microlitre. Probably the poorqualitative function of the lymphocytes in thischronically immunosupressed patientresulted in an opportunistic infection withCMV.

Conclusion: FBA is a descriptive fundusfinding and is not a disease entity. We needto accurately identify the cause of FBA toeffectively treat these patients. It would bewise not only to monitor the CD4 counts of

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Introduction:

World Health Organization committeedefining Occupational health says it shouldaim at the promotion and maintenance ofthe highest degree of physical, mental andsocial well being of workers in alloccupations; the prevention among workersof departures from health caused by theirworking conditions; the protection of workersin their employment from risks resulting fromfactors adverse to health; the placing andmaintenance of the worker in an occupationalenvironment adopted to his physiological andpsychological ability and to summarize theadaptation of work to man and of each manto his job (1).

There are various types of occupationswith varied complexity specific to individualjob. Eye care professionals like optometristsand ophthalmologists involved in visionscreening, would need, in the first instance,to know about the visual requirements of thejob the individual is engaged in, then planthe battery of vision tests based on visualrequirements of the job and finally suggestthe treatment to match the visual ability withvisual requirements /demand of the job. Thegoal is to minimize stress on the visualsystem, which will result in an efficient andsafe visual performance.

Occupational Vision Testing approach:

There are four important steps involvedin occupational vision testing. They are asfollows:

1) Occupational Analysis

2) Modified Clinical technique – usingconventional testing methods or usingvision testing instruments

3) Recommendation to match visual abilityto visual demand of the job

4) Follow-up – every year

1) Occupational analysis:

This is the first step in occupationalvision testing. There must be an analysis ofthe visual tasks involved in the occupationconcerned. The analysis requires theexaminer/optometrist visiting the occupationsite/job site and note down the factors suchas distance and size of the critical details ofthe task, color of the reference object, needfor depth perception, body, head and eyemovements, contrast and illumination of thework area. The potential hazards in the workplace also can be noted. This analysis willhelp in the appropriate selection of visualfunction to be evaluated in the next step.The logical method for determining the visualfactors required for a particular task has beenproposed by Grundy (2) and is given as theguide below:

Based on the job analysis, the visualdemand of the task can be planned.

2) Modified Clinical Technique: Theevaluation can be done by qualifiedoptometrists using standard instrumen-tations who can assess all the visualfunctions based on the input from jobanalysis and compare with the visualdemand of the job. This technique includeshistory taking, refraction procedure, ocularmotility examination, anterior segment andposterior segment evaluation, amplitude ofaccommodation, binocular vision assess-ment, visual field, color vision, contrastsensitivity testing and so on. Based onthe evaluation, referrals or recommen-dations can be made. There are othertechniques using Instrument screenersand computer programs which do notrequire optometrists. The screenersusually have internal lightning and thetargets are mounted either on a rotarydrum or separately on cards, which changemanually or by remote control. There are

Occupational Vision testing – An IntroductionR. Krishna Kumar, Elite School of Optometry

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many different vision screeners in themarket and all are modified stereoscopes.The types of tests commonly included inthe instrument screeners are: Visual acuity– distance and near, heterophoria –horizontal and vertical at distance andnear, stereopsis, fusion, color vision, and

visual field. There are few screeners tomeasure contrast sensitivity function also.Keystone and TNO Titmus II vision testerare the commonly available instrumentvision screeners available in the market.Computer programs are available toevaluate visual acuity, search tasks,

Occupational analysis

S. No Name General description of the task

1 Working distances (a) Far (beyond 200 cm) (b) Intermediate (200-55cm) (c) Near (55-30cm) (d) Very near (less than 30 cm)

2 Size of detail a) Large (angular size of critical detail over 10’) (b) Medium (5-10’) (c) Small (3-5’) (d) Very small (2-3’) (e) Extremely small (1-2’) (f) Minute (< 1’)

3 Main working positions a) Sitting b) standing c) Moving d) Mixture

4 Size of working areas (in which critical vision is required)

a) Large b) medium c) Small

5 Head movements a) Side to side b) up and down c) Mixture

6 Direction of gaze a) Ahead b) Up c) down d) side e) Mixture

7 Changes in direction of gaze a) Frequent b) Occasional c) Seldom

8 Movement of the task a) Stationary b) Slow movement c) Fast

9 Potential danger a) High risk b) Medium risk c) Low risk

10 Special accuracy or care a) Required b) Limited requirements c) not required

11 Binocular vision and stereoscope a) required b) not important c) Monocular vision adequate

12 Color vision requirement a) Good color discrimination required b) limited requirements acceptable c) Not required

13 Visual fields a) good field required b)Fair field acceptable c) Not important

14 Visibility (the relationship between the size of the detail, working distances, contrast and time available for viewing the task)

a) Good b) Fair c) Poor

15 Type of lightning in use, its adequacy and suitability

16 Eye protection requirements a) Required b) Not required

17 Hazards Basic, impact, Molten metal, dust, gas, chemicals, radiation laser, other

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oculomotor balance, fixation disparity andcentral visual fields (3,4).

3) Recommendation: Based on theexamination findings, the recommendationcan be either referral to appropriatespecialists or optical management in theform of appropriate lenses – correctingrefractive error with safety lens materials(polycarbonate, CR 39), useable color tint/coating and / or side shield, and orthopticexercises. In addition, appropriatemagnifiers and appropriate illuminationspecific for working needs will be advised.In rare situation, recommendation can begiven to the employee to switch over toanother visually less demanding jobappropriate to his visual capability.

4) Follow up: Every year evaluation of thevisual function has to be done to makesure that visual demand of the job andindividual visual capability continues tomatch.

In conclusion, for maximum visualperformance, the task and the worker shouldbe assessed so as to match the requirements

of the task with the visual capabilities of theworker.

Acknowledgement:

Dr P. P. Santanam, Faculty,Occupational Optometry of Elite School ofoptometry for introducing me to thiswonderful field of optometry.

References:

1) Jeanne Mager Stellman (1998),Encyclopaedia of occupational Health andSafety, 4th edition, International LabourOrganisation

2) Grundy J.W (1987), A diagrammaticapproach to occupational optometry andillumination. Optom Today, Aug 1,503-508

3) Rachel V.North (2001), Work and the eye,First edition, Oxford university Press, pp-1-23

4) Class room handouts of Dr.Santanam ourex-principal and professor of occupationaloptometry of Elite school of optometry,Chennai.

DNB REVISION COURSE 2005

JULY 7TH to JULY 13TH

CONDUCTED BY SANKARA NETHRALAYA CHENNAI

OSCE, CLINICS AND VIVA-VOCE

COURSE FEE: Rs.5000/-

Please Contact :Mr. N. SivakumarAcademic Officer

Medical Research Foundation18 College RoadChennai 600 006

Ph: 044-28271616, 28272823Fax: 044-28254180

E-mail ID: [email protected]

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The knowledge of Vitamin A – thechemistry, metabolism and deficiencyconsequences occurred rapidly in the firsteight decades of the 20th century.Identification of the intestinal beta-carotenecleavage enzyme in the laboratory of JamesAllen Olson paved way for understanding themechanism of formation of vitamin A fromingested carotenoids and studied dosedependent methodology clarifying thesequence of appearance of deficiency signsand symptoms. The synthesis of vitamin Aaround 1930 permitted fortification of foodsand supplements virtually eliminating theocular forms of vitamin A deficiency (VAD)as a public health problem in the developedcountries1. But it is still a fact that this hasnot happened even today in many developingcountries including India as VAD continuesto take quite a heavy toll on the sight andlife of children.

Vitamin A deficiency (VAD) is theleading cause of preventable blindness inchildren and raises the risk of disease anddeath from severe infections, especially inAfrica, South and East Asia. WHO sponsoredthe first systematic attempt to quantify themagnitude of the prevalence in the 1960s. In1995, WHO redefined VAD to include tissuedepletion below which functional impairmentwas likely. Serum level of less than 0.7µmol/L of vitamin A is used as the populationbased indicator of health risks . Nearly 75-140 million children of preschool age arevitamin A deficient and nearly one third ofthem become blind every year. Roughly 45%of VAD and xerophthalmic children andpregnant women with low-to deficient VADstatus live in South and Southeast Asia.These regions harbor 60% of all cases ofmaternal Xeropthalmia i.e. Nightblindness,

three fourths of whom seem to live in India 2.

Lack of vitamin A is particularly severefor preschool aged children once they havebeen weaned and are no longer dependenton breast milk as the primary source ofmicronutrients such as vitamin A, causingsevere visual impairment as well assignificantly increasing the morbidity andmortality from common childhood infectionsas diarrhoeal disease and measles 3. Forpregnant women in high-risk areas, vitaminA deficiency occurs especially during the lasttrimester when demand by both the unbornchild and the mother is highest. VAD mayalso be associated with mother-to-child HIVtransmission4.

Clinical VAD Surveys — UNICEF (Year, 2000)

Area Sample Size

Indicator/ Prevalence

(%)

India MICS 2000 prevalence of Night Blindness 0-4 yrs

65,741 0.6%

ICMR ,District Nutrition Project Survey 2001 XN

6633 2.8%

NINWHO/UNICEF Orissa & AP study using serum retinol <0.70umg/l as a cutoff 12-36 months base line and post VA

2,134 Orissa

AP 2,120

Orissa 63.8%

AP 52. 3%

ICMR 16 district study Night blindness pregnant women in India (2001) Night blindness among children 24-71 months

6,633

113,202

2.7%

1.03 %

Dried Blood Spot (DBS) for Vitamin A analysis– a field friendly methodN. Angayarkanni, A. V. Sai Jyothi, K. Coral, R. Punitham, K. Krishnakumar, Madhava Rao andS. Ramakrishanan

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Vitamin A and carotenoids 5: Out of thehundreds of carotenoids in nature only asmall number are present in our food andthese are of two kinds. One is beta-carotene,the most important and the one that can beconverted in the body to retinol. It is split byan enzyme in the intestine to produce 2molecules of retinol. The other kind ofcarotenoid including lutein and lycopene etc.cannot form vitamin A, though they haveother important functions. Laboratorymethods are now available for measuring theamount of different forms of retinolcollectively known as retinoids. The HPLC(high performance liquid chromatography) hasreplaced most other methods of vitamin Aestimation.

Mechanism of action of vitamin A 5:Like lipids, retinol through the small intestinebecomes part of chylomicrons that enter theblood circulation reaching liver and is storedin special cells called stellate cells as retinylesters. The transport to the other parts ofthe body is as retinol through the retinolbinding protein (RBP) as well astransthyretin (TTR). At the cell membraneretinol is taken up by receptors (RBPreceptors) and within the cell retinol bindsto the cellular retinoid binding proteins(CRBP) synthesized independently. Mostimportantly it is also directed to the nucleusof the cell. The nuclear receptors (RAR andRXRs) are activated by the acid form of theretinol called the retinoic acid, which is theactive form in the tissues except the eye.Many genes are activated by these receptorsand they function like hormones such as thesteroid with which they are closely linked.The functions of vitamin A are brought aboutby this mechanism except for the mechanismof vision. This is because the functional formof vitamin A in the rods and the cone cellsis the 11-cis retinaldehyde (retinal) and notretinoic acid. Thus the vitamin A functionsare mediated through the two oxidizedmetabolites of retinol namely retinaldehydeand retinoic acid.

Test for vitamin A deficiency 5: Whileit is encouraging to note that the rate of

blindness due to Xeropthalmia is steadilydecreasing, only in recent years has it becomeevident how widespread sub-clinicaldeficiency is. The majority is from the southand south east Asia (WHO, 2000). Of greatimportance is the fact that only carrying outbiochemical or other laboratory tests canreveal the deficiency. This sub-clinicaldeficiency is much more common andwidespread than clinical deficiency likexerophthalmia affecting young children andmothers who are either pregnant or lactating6.

Sub-clinicalØ Reduced storesØ Lowering serum levelØ Metaplasia

Clinical↑ deficiency Ø Xeropthalmia

The state of vitamin A nutrition in thebody is called vitamin A status. Thebiochemical test measuring serum retinol isthe test that has been most widely used. WithHPLC accurate estimates can be made.However, the level of retinol in serum doesnot truly reflect changes in vitamin A statusbecause it is in equilibrium with the storesin the liver and some other organs. Serumretinol does not fall appreciably until bodystores have been virtually exhausted. Eventhen serum retinol values for large populationgroups have been found to be reliable of theiroverall vitamin A status. Presentlymeasurement of serum retinol binding protein(RBP) is gaining importance. The vitamin Astatus is sometimes assessed also byconjunctival impression cytology. Both theWorld Health Organization and UnitedNations Children Fund have proposed the useof serum retinol as a key indicator of vitaminA deficiency.

Table:

Ranges of prevalence of serum retinolto define a public health problem of sub-clincial vitamin A deficiency in youngchildren5.

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Presently a more appropriate cut off of0.50 µmol/L instead of 0.70 µmol/L isproposed for marginal deficiency 5.

Use of Dried blood spots – a field method ofcollecting blood samples

Currently we have switched over to themost frequently used method for measuringvitamin A status using HPLC from thespectrophotometric method. The method isto take venous blood samples, centrifugethem (and after freezing and transporting tothe lab if necessary) to measure the retinolcontent in serum by HPLC. This is quitetedious as well as expensive in terms ofcollection and transport for field collections.A less expensive and more efficient way is tomeasure Retinol Binding protein (RBP), whichcorrelates well with the retinol content is theELISA technique for measuring RBP alongwith C-Reactive protein (CRP) and alpha-1glycoprotein (AGP) as indicators of acute andchronic infections 7.

In developing countries collecting venousblood in field studies particularly in childrenis not very easy. The cultural beliefs, localtaboos, use of needles as well as the needfor trained phlebotomist and the risk oftransmitting blood-borne viral diseases insuch field studies complicates the study.Moreover isolation of the serum component,storage and transport are the other obstacles.

But recently Craft Technologiesdeveloped a procedure to measure bloodretinol concentration in venous or capillaryblood from dried blood spots (DBS). They alsovalidated the method by comparing it withvenous and capillary serum retinol andconcluded that it is possible to compare bloodretinol concentration from DBS 8,9,10 .

The objective of this study was to adoptthe same method, suitably modified, in orderto compare it with the existing method ofvitamin A (retinol) estimation in venous blood

by HPLC with that of the DBS specimen andto arrive at the recovery if retinol in the DBSmethod, so that it can be adopted in thefuture field studies.

Subjects and Methods:Two blood specimens were collected, for

dried blood spots as well as serumseparation. 4 samples for retinol analysisbased on the method of Craft et.al 8 weregenerated. The subjects were either in thefasting or the postprandial state. 1-2 dropsof the venous blood (40 µL) was placed usinga micropipette, onto each of the bloodcollection spot on the card. The remainingwhole blood was allowed to coagulate at roomtemperature (25°C) and centrifuged at 1500X g and the serum transferred to a vial. Carewas taken to protect the samples from lightduring all procedures. The DBS cards wereallowed to dry in the dark (in a cupboard)for 1 hr. The cards were then stored in ziplogbags at –20°C for a maximum of 2 weeks.

Materials : Retinol, Retinyl acetate,Hydroquinone, Butylated hydroxytoluene,(sigma) ascorbic acid, diethylene triaminepenta acetic acid, (Merck) methanol, ethanolhexane, acetonitrile (Merck, HPLC grade) andDBS cards (Schelicher and Schuell 903specimen collection paper, Germany )

Extraction of retinol7: The total 13.0 mmdiameter circle on the card was cut from theDBS card (Fig 1) and extracted in 1.0ml of0.15M phosphate buffer, containing 60mMascorbic acid as an antioxidant and 2mMDTPA, sonicated for 5 minutes andcyclomixed for 10 minutes. Subsequently0.9ml of ethanol containing 100mM BHT wasadded. Retinyl acetate (10 µg) was used asthe internal standard and vortexed for 20seconds. One ml of hexane was added andcyclomixed for 1 minute for extraction andcentrifuged for 1 minute at 1500xg. Thehexane layer was removed and the extraction

Indicator Mild Moderate Severe

Serum retinol >2 — <10% >10 — < 20% >20%

(< 0.70µmol/L)

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and water, 65:34:1), cyclomixed for 1 minuteand 50µl of the sample was injected into theHPLC (HP-HPLC). The HPLC separation ofretinol was performed isocratically usingacetonitrile and methanol and water (65:34:1)at a flow rate of 0.5ml/min in a hyper-siloctadecyl silane column (C18, 5 µ ). Retinolwas monitored at 325 nm using the variablewavelength UV detector.

Results:

Linear calibrations were prepared using4 concentrations of retinol as standard(20,40,60 and 80ng.concentrations) for theestimation of retinol in the serum and DBSsamples.

S.No. Name

Serum retinol (venou)s (µg/dl)

DBS retinol (µg/dl)

1 Control 1 32.8 28.8

2 Control 2 30.2 25.2

Ø Plasma volume adjustment factor = Serum

retinol / DBS retinol = 1.2

Ø mean of ± 11% variation

Fig 2 shows the chromatogram ofstandard retinol(RT=4.01min). Fig 3 showsthe serum retinol with a retention time of4.1min. Fig 4 shows the DBS extracted retinol(RT=4.07min) while Fig 5 shows DBS spikedwith external (10µg retinol) and the internalstandard (10 µg retinyl acetate). Retinylacetate had a retention time of 5.1 min.

NAME__________________________

DATE__________________________

S&S®903TMLot#WO11

NAME__________________________

DATE__________________________

S&S®903TMLot#WO11

Fig 1: DBS Card

min0 1 2 3 4 5 6 7 8 9

mAU

0

0.5

1

1.5

2

2.5

3

3.5

4

VWD1 A, Wavelength=325 nm (VITAMIN\AA000346.D)

4.0

12 -

Vit

A

Fig 2: Retinol Standard (20ng)

min0 1 2 3 4 5 6 7 8 9

mAU

0

2

4

6

8

10

12

14

VWD1 A, Wavelength=325 nm (VITAMIN\AA000379.D)

2.7

45

4.1

61

Fig 3: Serum retinol

min0 1 2 3 4 5 6 7 8 9

mAU

0

0.5

1

1.5

2

2.5

VWD1 A, Wavelength=325 nm (VITAMIN\AA000376.D)

2.6

98 3

.080

3.1

49

Fig 4: DBS retinol

process repeated. The hexane layers werepooled, and dried under a stream of nitrogenand the residue obtained was dissolved in200µ l mobile phase (methanol, acetonitrile

min0 1 2 3 4 5 6 7 8 9

mAU

0

2

4

6

8

VWD1 A, Wavelength=325 nm (VITAMIN\AA000373.D)

2.6

91 3

.106

3.1

82

4.0

86 -

V

it A

Fig 5: DBS spiked with external (10ugretinol) and Internal standard (10ug

retinyl acetate)

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Discussion:

Quite often it is impossible to obtainvenous blood samples from specificpopulations. A finger prick method ofobtaining the sample needs to be adopted asa smaller volume is involved. The approachof collecting and drying the small bloodsamples on filter paper for further extractionand analysis off the site of collection becomesmore valid in the context of not only in thetransport of specimen over larger distancesbefore analysis but also eliminates the needfor even small machineries as well as theneed for expertise to collect and process thesample at the site of collection.

More number of samples is beinganalyzed by the DBS method andsimultaneously in the venous blood forvalidation of the methodology. The majoradvantage of using the entire spot is thathigher sensitivity is obtained and unevendistribution of retinol throughout the wholespot is avoided. Using the DBS method wewere able to get reproducible results thatwere comparable to our previous extractionprocedure from the serum followed by thereverse-phase HPLC separation, with thesame retention time of 4.1 min and a meanvariation around 11%.

Retinol being bound to retinol bindingprotein is greatly protected from degradationand remains stable even at room temperature.Erhardt et.al have reported that DBS is stablefor 3 months and longer when samples arestored at about 23oC in the dark. But still itis preferable to maintain the sample at coolertemperature if possible. Of the storageconditions tested as reported by the samegroup, it is reported that the storage ofinadequately dried DBS in a Ziploc bagwithout desiccant was detrimental to DBSretinol analysis.

Thus this method once validated in ourlaboratory with more number of samples canbe used in larger studies where plasma orserum samples are not readily obtained. Butthe sensitivity of the method to detect severedeficiency as against the serum retinol is yetto be reported.

Reference:1. Underwood BA. Functions and actions of

retinoids and carotenoids: Building on thevision of James Allen Olson: Vitamin Adeficiency disorders: International effortsto control a preventable “Pox”. AmericanSociety for Nutritional Sciences. 231S-234S.

2. Keith P. West. Extent of Vitamin ADeficiency among Preschool Children andWomen of Reproductive Age1,2.Proceedings of the XX InternationalVitamin A Consultative Group MeetingJ. Nutr. 132: 2857S–2866S, 2002.

3. Yamamura,CM, Sullivan,KM, Frits van derHaar, Auerbach SB, Iohp,KK. Risk factorsfor vitamin A deficiency among preschoolaged children in Pohnpei, Federal statesof Micronesia, J.Tropical Pediatrics . ,2004,50: 16-19

4. Véronique Azaïs-Braesco and GérardPascal Vitamin A in pregnancy: require-ments and safety limits, Am J Clin Nutr2000;71(suppl):1325S–33S.

5. McLaren DS, Frigg M. Sight and LifeGuidebook on Vitamin A in Health anddisease. 2nd ed. 2001.

6. Thurnham DI, McCabe GP,Northropclewes CA, Nestel P. Effects ofsubclincal infection on plasm retinolconcentrations and assessment ofprevalence of vitamin A deficiency : metaanalysis. Lancet 2003;362 (9401):2052-8.

7. Erhardt J, Tropmed S. Use of dried bloodspot (DBS)- A simple and field-friendlymethod of collecting blood samples for themeasurement of vitamin A status. Sightand Life Newsletter1/2005; 4-6. .

8. Craft NE. Bulux J, Valdez C, Yukang Li,Solomons NW. Retinol concentrations incapillary dried blood spots from healthyvolunteers: method validation. Am J ClinNutr 2000;72:450-4

9. Craft NE, Haitema T, Brindle LK, YaminiS, Humphrey JH, West KP. Retinolanalysis in dried blood spot by HPLC .J.Nut. 2000, 130: 882-885.

10. Erhardt J, Craft NE, Heinrich F andBiesaiski HK. Rapid and simple measure-ment of retinol in human dried wholeblood spots. 2001;318-21.

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35 years old man presented with double vision in up and down gaze, with history of injurywith a stick one month back.On examination visual acuity was 6/6 N6 OUwith RAPD 2+ in left eye.

Anterior and posterior segment examination is with normal limit except for sutures of upperlid tear repair with mild edema.Ocular motility is shown in the photos. What is your diagnosis?

(Answer in next page)

Muscle PuzzleAnand Vaijwade and S. Meenakshi, Sankara Nethralaya ORBIS Pediatric Ophthalmology Learning andTraining Centre

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(Answer to Muzzle Puzzle in page 25)

Extra ocular movement shows limitation of up and down gaze in left eye. Cover testshows ortho in primary gaze with 25 PD left hypotropia in up and down gaze, 15 PDexotropia with flick right hypertropia in right gaze and 4 PD esotropia in left gaze.

CT scan showed fracture of anterior, medial wall and floor on left side with entrapmentof Inferior rectus.

On Diplopia charting patient had binocular crossed vertical diplopia more in down andup gaze. Hess charting showed under action of Inferior rectus.

Forced duction testing was positive for inferior rectus restriction.

Strabismus after blow out fracture has been estimated to occur in about 58 % ofpatient. The term “blow out fracture’’ was coined by Smith and Regan. These are seen afterinjury with cricket or tennis ball or road side accidents. Immediately following injury itpresent as black eye and restriction of ocular movement in all gazes which usually subsidesby end of first week and presents as specific restrictive strabismus.

Patient develops hypoesthesia along infra orbital nerve, diplopia in up and down gazedue to entrapment of soft tissue in fractured fragment and enophthalmos due to herniationof orbital contents in to the maxillary sinus.

The presence of muscle entrapment can be confirmed on FDT (force duction test).Saccadic movement testing can differentiate between paretic and entrapped muscle. Usuallyassociated severe globe damage is rare since blow out is a protective phenomenon.

Plain X ray (Waters view) and CT scan aid diagnosis.

In initial phase of injury, surgery should be withheld until two weeks till edema subsides.Patient should be followed up closely with serial diplopia and Hess charting .

Surgery should be defered till sufficient diplopia free area is present in primary anddown gaze. Surgery can also be performed for significant enophthalmos. Teflon plate can beplaced subperiostially. For troublesome diplopia fresnel prisms trial can be considered. Forsurgical correction first inferior rectus recession can be planned.

This issue of Insight is sponsored by:Rampion Eyetech Pvt. Ltd., Kalash, New Sharda Mandir Road, Paldi, Ahmedabad - 380 007Apex Laboratories Pvt. Ltd ., 44, Gandhi Mandapam Road, Kotturpuram, Chennai - 600 085Pharmacia & Upjohn India Pvt. Ltd., SCO 27, Sector 14, Gurgaon 122 001, HaryanaWarren Pharmaceuticals Ltd., 2/10, Joti Wire House, Veera Desai Road, Andheri (W), Mumbai - 400 053

Medical & Vision Research Foundations thank the above sponsors for their generosity.

Editor : Dr S Meenakshi 26 Private Circulation Only

INSIGHT & EYELIGHTS are now available on line athttp://www.sankaranethralaya.org/publication.htm

PLEASE LOOK US UP ON THE WEB athttp://www.sankaranethralaya.org

Please forward your correct address with your E-mail address if any,to enable us to update our address data base.

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G L A U C O M ACurrent imaging modalities have added animpetus to diagnose and detect glaucoma early.Emphasis to use these in a rational manner isimportant for high quality glaucoma management.There is a consensus that very little is knownabout the exact pathophysiology of glaucoma andresearch for evidence at molecular level isbecoming a priority “This possibly will be the wayto go”. Constant efforts to find the ideal surgicaltechnique have been yielding qualified results.Let’s gather and upgrade information on evidencebased management of glaucoma

OCULOPLASTYThe board sweep of oculoplastic surgeryencompasses the areas of orbital, eyelid, andlacrimal disease and has seen rapid strides andexciting developments in the recent past. Thereis a surge of interest in this area and this sub-specialty session aims to provide an exciting rangeof topics to be discussed by experts and of interestboth to the oculoplastic surgeon and the generalophthalmologist, including students in training.

VITREO RETINAThe program includes 4 mini-symposiums: VRsurgery and diabetic retinopathy update, age –related macular degeneration update, posteriorsegment complications of anterior segmentsurgery and anterior segment complications ofposterior segment surgery.Vitreoretinal surgery update would highlightnewer innovations such as 25 G instrumentations,role of newer imaging techniques such as OCT in

understanding vitreoretinal interface, newer dyesto stain ILM, pharmacological agents for macularedema, surgical approaches to manage venousocclusions. Diabetic retinopathy update wouldfocus on epidemiology, emerging trends, systemicconsiderations, and role of diabetic vitrectomy.

Age-related macular degeneration update wouldgive and overview of clinical profile, role ofancillary tests, polypoidal choroidal vasculopathy,role of TTT/PDT, pharmacologic agents andsubmacualar surgery.

Posterior segment complications of anteriorsegment surgery and vice versa would involveissue as globe perforation, posteriorly dislocatednucleus/IOL, cystoid macular edema.Suprachoroidal hemorrhage, and managementissues related to muscle imbalance, cornea,cataract and glaucoma following vitreous surgery.

CATARACTThe cataract sessions will deal with small incisioncataract surgery. Instruction courses on thepopular small incision techniques are planned.Prof Michael Blumenthal, one of the pioneers ofmanual small incision cataract surgery, willconduct an instruction course on the Blumenthaltechnique of cataract surgery. These sessions willdeal with all aspects of the techniques includingwound construction, instrumentation and nucleusmanagement. Doing cataract surgery in difficultsituations with each of these methods will alsobe highlighted. The meeting has sessions devotedexclusively to the management and avoidance ofcomplications.

Sankara NethralayaInvites you to

Indo Israel Ophthalmic CongressNovember 19 & 20, 2005 - Chennai

Dear Colleague,

I have great pleasure in inviting you for the inaugural Indo Israel conference. This meetingwill bring together luminaries from India and Israel under one roof.

Over the two days there will be sessions addressed by experts from the premier institutionsthe highlight being the session on cataract surgery by Prof Blumenthal and team. Othertopics to be covered include updates on the latest trends in glaucoma, oculoplasty, diabeticretinopathy and ARMD. The program will feature a plenary session addressed by Internationalleaders in Ophthalmology. Sessions for free posters are also planned.

In addition to high quality scientific discussions an evening of cultural events and a galadinner will give you the opportunity to interact with the faculty.So don’t miss this opportunity to participate in this unique program.

I hope to see you in Chennai.Dr Prema Padmanabhan.

Scientific Programme

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PatronsProf. M BlumenthalDr. S S Badrinath

List of Invited FacultyDr. Anat Lowenstein, IsraelDr. Aung Tin, SingaporeDr. Ayala Pollack, IsraelDr. Dov Weinberger, IsraelDr. Ehud Assia, IsraelDr. Elish Bartov, IsraelDr. Issac Hemo, IsraelDr. Joseph Moisseiev, IsraelDr. Melamed, IsraelDr. Nachum Rosen, IsraelDr. Pe’er J. Israel

andfaculty from host institutions

Registration DetailsFees

Delegate Type Up to Regular Spot31.07.05

Indian Rs.1000 Rs.1500 Rs.2000Post GraduateStudents,Indian Rs.750 Rs.750 Rs.1000InternationalDelegates USD 200 USD 200 USD 200

Poster Specification4 feet height, 3 feet width

(4/3 in vertical)Maximum No of PPT Slides - 18

HostSankara Nethralaya

18, College Road, Chennai – 600 026.Phone: 91-44-29271616

Co-HostsLV Prasad Eye Institute, HyderabadAravind Eye Hospital, MaduraiSankaradeva Nethralaya, GuwahatiShri Ganapati Netralaya, Jalna

Congress Secretariat

Ms J Revathy, Sankara Nethralaya - JKCN Complex,21, Pycrofts Garden Road, Chennai 600 006.

Tel.: 91-44-28233556, 28271616, 28311913 Fax: 91-44-28254180Email: [email protected] Website: www.i2ioc2005.org

Registration Form

Name : …………………………………………………………………………………………………………………………………………

Institution : ………………………………………………………………………………………………………………………………

Address : ……………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………………

Tel : …………………....………………………......... Email : ……………………….........………………………………….........

Please find enclosed herewith my cheque / DD No. ………………………….. Dt. ……………………………

For Rs. / USD ……………………….........………………………......... towards registration fee for

Delegates. Cheque / DD should be drawn in favour of “Medical Research Foundation”

Chennai.

Date

Place Signature

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