Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

Users Online : 65487

AbstractMaterial and MethodsResultsDiscussionAcknowledgementReferences
Article in PDF How to Cite Citation Manager Readers' Comments (0) Audio Visual Article Statistics Link to PUBMED Print this Article Send to a Friend
Advertisers Access Statistics Resources

Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Original article / research
Year : 2009 | Month : April | Volume : 3 | Issue : 2 | Page : 1381 - 1388

A Novel Approach To The Definitive Diagnosis Of Enzymopathies

APTE B. N. Correspondence Address :
DR. BALKRISHNA. N. APTE
Consultant Medical Geneticist and Project and Programme Director,
Vidyasagar Institute of Genetic Studies and Research,
Rail View, Opp.Swaminarayan Temple, Dadar (East), Mumbai – 400 014.
Tel. Clinic - 2410 5924, 2415, 2751 Res. -2515 1226
Mob. - 9821461434 Email - balnapte@rediffmail.com

Abstract

Enzyme estimations are invariably carried out using synthetic chromogenic or fluorogenic substrates, which are structurally very simple as compared to the natural substrates. A defective enzyme resulting from mutation in the structural gene loses its activity on the natural substrates, leading to a genetic disease. Such an enzyme, at times, may still retain substantial catalytic activity on the synthetic substrate. If the enzyme activity falls within the normal range, the situation poses a problem in the diagnosis of the patient. To circumvent this situation, we have invented a novel approach which has enabled us to arrive at an unambiguous, definitive diagnosis of patients. Our novel approach will be presented here.

Introduction
Mutations in the structural genes of enzymes lead to alterations in their catalytically active structure. These alterations in mutant enzymes result in their reduced (or no) activity on the synthetic as well as natural or in-vivo substrates (2), (8),(9),(10),(20). The deficiency of an enzyme results in a genetic disease. Each enzyme has a normal range of activity. For example, Arylsulphatase A (EC 3.1.6.1) is considered normal if its activity falls between 200 and 600 units (nmoles / mg protein / hour). A person with enzyme activity between 200 and 600 units will therefore, be considered normal. A carrier of the disease is also defined in terms of a range of enzyme activity. For Arylsulphatase A, a carrier will be one whose enzyme activity falls between, let us say, 50 and 200 units. Any individual showing activity below 50 units will be considered as a patient. All of these ranges of activities used for defining a patient or a carrier or a normal individual are conventional and are solely based on the experience and the data of different laboratories over a number of years. There are no sharp cut-offs between the three categories of individuals. At times, the patients may have enzyme activities extending into the carrier range or even beyond into the normal range. Indeed, cases have been documented where a patient showing fulminant clinical manifestations of Metachromatic Leukodystrophy (MLD) has normal Arylsulphatase A activity (15). Cases have also been documented, where an individual with a zero Arylsulphatase A activity is normal on all counts (5).

In all these methods of diagnosis of enzymopathies in the prior art, the enzyme estimations are done by using synthetic chromogenic or fluorogenic substrates. A single point enzyme activity measurement is made at the end of the incubation period. The diagnosis of the patient is based solely on this single point estimation.

We present here a novel approach, henceforth referred to as Apte procedure (1), to circumvent this problem of ambiguity in the diagnosis. Our invention led to a differential and definitive diagnosis of the patient, the carrier and the normal individual.

Material and Methods

Leukocyte Enzyme Estimation
Blood samples (10ml) were collected in heparinised bulbs from the patient, the patient’s father and mother as carriers, and a normal individual to serve as a control. Leukocytes were obtained from each of these samples by following the procedure described by Hans Galjaard (7). Leukocyte lysates were prepared by sonication. The sonicates were centrifuged to get clear supernatants which were used for quantitative enzyme estimations using the appropriate chromogenic or fluorogenic substrate for each enzyme.

Urinary Enzyme Estimation
About 10 ml of midstream urine samples were collected from the patient, the patient’s father and mother as carriers and a normal person. 5 ml from each urine sample was dialyzed against distilled water for 18 hours in cold. The dialyzed urine samples were centrifuged at 5000 rpm for 10 minutes to remove any suspended matter, after which they were used for the quantitative estimation of the enzyme.

Serum Enzyme Estimation
Blood samples (3ml) were collected in plain bulbs containing no anticoagulant. The blood was allowed to clot. The tubes were centrifuged at 3000 rpm and the clear supernatant serum was collected and used for the estimation of enzyme.

We describe below, the application of the Apte procedure to the study of one leukocyte and urinary Arylsulphatase A (EC 3.1.6.8) (4),(11),(12) and one enzyme in serum, Biotinidase (EC 3.5.1.12), as illustrative examples. All the enzymes were assayed according to the published procedures (7). Arylsulphatase A was assayed by using p-nitrocatechol sulphate as the synthetic, chromogenic substrate (15), (7), (11). Biotinidase was assayed using biotin-4-amidobenzoic acid as synthetic chromogenic substrate (18), (19). All enzyme estimations were carried out at 370 C and one-point readings were taken at the end of the incubation period. The leukocyte lysates, the urine and the serum samples were stored at –100C after enzyme estimations. The enzyme estimations on the same samples were repeated every 24 hours for the next three days. The percentage of enzyme activity retained was calculated for each day, assuming the enzyme activity in each sample on the first day to be 100 %.

Results

Studies on one leukocyte enzyme, one urinary enzyme and one serum enzyme are presented here as illustrative examples.

Example 1
Deficiency of Arylsulphatase A causes Metachromatic Leukodystrophy (MLD, Mckusick 250100) in which the patient suffers from gait disturbance, mental regression, peripheral neuropathy, progressive dementia and muscle weakness. The enzyme was quantitatively estimated in the leukocytes of a 2-year-old patient (P-1), his father (FP-1) and mother (MP-1) as carriers and 2-year-old normal child (NP-1), as described in the materials and methods. The enzyme activity was followed up for three consecutive days. The percentage of the enzyme activity retained on each day was calculated, assuming the enzyme activity on the first day of estimation to be 100%. The results of the enzyme estimations in all these samples for Arylsulphatase A are shown in (Table/Fig 1) and graphically in (Table/Fig 2).

Example II
Deficiency of Arylsulphatase A causes MLD, as described before. This enzyme can also be measured in urine (4). Urine samples of a 2-year-old patient P-2 and the patient’s father FP-2 and mother MP-2 as carriers and a normal 2-year-old child NP-2 were collected and processed as described in the materials and methods. The enzyme activity was followed up for three consecutive days and the percentage of the enzyme activity retained on each day was calculated, assuming the activity on the first day to be 100%. The results of the enzyme estimations in all of these urine samples for Arylsulphatase A are shown in (Table/Fig 3) and graphically in (Table/Fig 4).

Example III
Defective Biotinidase activity causes Multiple Carboxylase Deficiency (McKusick 253260). This results in ataxia, muscular hypotonia, seizures, alopecia, periorificial cutaneous eruptions, and episodic metabolic acidosis. The enzyme Biotinidase was estimated in serum. Blood sera of a 6-month-old patient P – 3, the patient’s father FP –3 and mother MP – 3NP – 3 were collected as described in the materials and methods. The enzyme activity was followed up for three consecutive days and the percentage of the activity retained on each day was calculated, assuming the activity on the first day of assay to be 100 %. The results of the Biotinidase estimation in all the samples are shown in (Table/Fig 5)and graphically in (Table/Fig 6).

Example IV
We now describe a case of a 56-year-old male patient HSB, whose Arylsulphatase A activity was 400 units, which was well within the normal limits. However, the patient exhibited a classical picture of MLD with fulminant clinical manifestations such as ataxia, muscular hypotonia, dementia, etc. All of these clinical manifestations were progressive and had started at the age of 52 years. HSB was investigated before he came to us for various possible enzymopathies. However, he was not given any definitive diagnosis. We decided to apply the Apte procedure to measure the Arylsulphatase A activity in his leukocytes as well as in his urine sample. Leukocytes and urine sample from a normal person, NP-4, of the same age were also run together with samples as controls. The results of this experiment are shown in (Table/Fig 7) and graphically in (Table/Fig 8)

Discussion

(Table/Fig 1) shows the decay of Arylsulphatase A, over a period of four days. The day on which the enzyme activity is measured first, was the first day. Arylsulphatase A activity came down to 76 % in the normal person (NP-1) on the fourth day, whereas the activity of this enzyme in the patient (P-1) went down to zero on the second day itself. The enzyme activities in the patient’s father (FP-1) and mother (MP-1) who are carriers of the disease, became zero on the third day.

The example of the decay of Arylsulphatase A in urine shown in (Table/Fig 3) and (Table/Fig 4) is interesting, as it follows the same pattern of decay as that in the leukocytes. Normal subject, NP-2, retained 63% of his original enzyme activity as against the patient, P-2, who retains only 5%. The patient’s father and mother, FP-2 and MP-2 respectively, retained 35% and 32% of their initial enzyme activity on the 4th day.

The example of the decay of Biotinidase is of particular interest. The enzyme activities in both carriers of the disease FP-3 and , , the father and the mother, were lower than the patient, P-3, to start with. However, the patient retained only 3% of his initial enzyme activity on the fourth day of the assay, whereas the patient’s father and mother retained 36% and 34% of their initial enzyme activity, respectively. The normal person, NP-3, retained 69% of his initial Biotinidase activity.

Another interesting observation made, was in the decay of leukocytes and urinary Arylsulphatase A in the patient, HSB, and the normal person. The patient, HSB, had a much higher leukocyte Arylsulphatase A activity of 400 units, as compared to the corresponding control, NP-4, who had 256 units. The patient’s urinary Arylsulphatase A was in the carrier range (3.8 units). The rate of decay of the leukocyte enzyme of the patient followed hand in hand with that of the urinary enzyme. The rates of decay of the leukocytes and the urinary Arylsulphatase A in the normal person were much lower than that of HSB.

The patient retained only 23% of the initial enzyme activity on the fourth day of the assay, whereas the normal person retained 66%, thus allowing us to differentiate and diagnose the patient with certainty. This problem had precluded confirmed diagnosis for HSB before we applied the Apte procedure to this case.

We carried out another interesting experiment to ascertain the validity of the Apte procedure. We had a patient, MS, who started showing the clinical manifestations of MLD at 46 years of age. The decay kinetics of his leukocyte and the urinary Arylsulphatase A, confirmed the diagnosis. He has a daughter. His wife was found to be unaffected for MLD, with her urinary Arylsulphatase A activity of 7.4 units with the decay kinetics, corresponding to that of a normal person. Thus, MS was a patient and his wife was normal. Therefore, their daughter was suspected to be a carrier. The urine samples of MS, his wife BS and their daughter DS were collected and processed as described in the materials and methods. The decay of Arylsulphatase A in these urine samples was followed up and is shown in (Table/Fig 9) and graphically in (Table/Fig 10). The kinetics of the decay of the enzyme of the patient’s daughter, DB, falls in between that of her father and the mother, thus confirming the diagnosis of DB as a carrier.

The Apte procedure was successfully applied to the prenatal diagnosis of a case of suspected MLD. The previous sibling had died with MLD at 3 years of age. The enzyme in the chorionic villi showed a normal rate of enzyme decay. Both parents showed the decay of the enzyme activity, which was consistent with their carrier status. A healthy child was born to the couple who is now a 12 year old, school going boy.

The question here is, why the enzymes in the patient, the carriers and the normal person showed different rates of decay of activity?

The amino acid sequence of an enzyme is the unique determinant of the three dimensional structure and, therefore, of its active state. A change of an amino acid due to a missence mutation will alter the structure and hence affect the activity. Such a structure will be fragile and will fall apart quite easily. It is also known that denatured proteins are excellent substrates for proteolytic enzymes. An enzyme with an altered, fragile structure will also form a better substrate for proteolysis than the enzyme in its natural, native state.

Studies on the inactivation of the highly purified bacterial α-galactosidase enzyme at the single molecular level have shown that thermal denaturation of the enzyme resulted in an all-or-none inactivation (13). In contrast to this observation, it is shown that enzyme inactivation caused by prolonged storage, results in a population of molecules with intermediate levels of activity ranging from five to one hundred percent of that present in the native Beta-galactosidase molecule (14). One of the hypotheses proposed for this observation was that the molecules of the aged enzyme are in a state of lower total free energy than those of the freshly synthesized enzyme. This implies that different active conformational states can be obtained with identical amino acid sequences. The rapid loss of enzyme activity in our experiments could be explained partially by this hypothesis, provided, it is proved that the number of intact molecules of the enzyme remain constant even on the fourth day.

Experiments on Beta-galactosidase were done with highly purified, homogeneous preparations. Our system is crude. It consists of the total leukocyte extract or the urine or the serum containing myriads of other components, including a large number of proteolytic enzymes. It is therefore, highly likely, that the loss of enzyme activity is due to the physical loss of the enzyme molecules and not due to their conformational alterations. The patient’s enzyme would have had the most fragile structure, which would have made it most amenable to the proteolytic attack. The normal person would have had a tighter, native enzyme structure, making it less accessible to proteolysis.

All of the enzymes under the present study are multimeric and had a quaternary structure. Therefore, various possibilities of association of different subunits – native and the mutant ones – in carriers makes the picture even more complex (6). If the hybrid structure assumed normal conformation, it would behave like the native enzyme. The decay of these molecules would be like those of the native enzyme. Reduced rates of activity decay in carriers could well be explained on this basis. Structural studies on the enzymes in the normal person, the carriers and the patient will clinch this issue.

On the contrary, there may be a large number of other epigenetic factors, which would go to make an active enzyme structure. In Beta-Galactosidase for instance, there are at least two other factors, which are involved in shaping the final catalytic structure of the enzyme. A glycoprotein called Cathepsin A and a substance called Saposin B. Glycoproteins stabilize Beta-Galactosidase. The same glycoprotein can activate Neuraminidase in the Lysosomes (3), (16),(17). Saposin B, on the other hand, can act as a physiological detergent, facilitating the interaction of the substrate and the enzyme. One can consider defects in the structure of these two additional entities, which would result in the defective Beta-Galactosidase. There are many such examples in the human enzyme systems and they should be kept in mind while formulating a model for the decay of enzyme activities when stored at –100C. Then there are chaperoning proteins, which guide the enzymes to their appropriate destination. The hydrolases will be guided to the lysosomes. The oxidases and peroxidases will be guided to the peroxisomes. Any mistake in the tag of the enzyme will result in the enzyme(s) not reaching the final destination, or its leakage out of the cell. This can affect the ‘stability’ of the enzyme in terms of its activity, as measured by the conventional procedure. We should keep all these options open when we think of the ‘stability’ of the enzyme.

We have extended our studies of the application of the Apte procedure to several other enzyme systems such as, α-Galactosidase, α-Iduronidase, Arylsulphatase B, Beta-Glucuronidase, etc, and in all cases, the procedure has enabled us to differentially diagnose the patients, the carriers and the normal persons without any ambiguity. The most satisfying part of the procedure is the generality of the application of the procedure to any pathological sample and to any enzyme system. Let us say that Arylsulphatase A is well within normal limits in the leukocytes of a patient. It may be in the carrier range in his plasma and may be in a range, which raises suspicion about the individual as to whether he is a patient. When the decay kinetics of the enzyme is carried out in all his samples, the rate of decay in all the samples is exactly identical, thus providing an unambiguous result and hence a definitive diagnosis.

The generality of the application of the Apte procedure to the study of enzymes in any pathological sample makes it highly appealing. The clear-cut results given by the procedure put the test subject under one of the three categories- a patient or a carrier or a normal person. One of the drawbacks of the procedure, as we saw, was the sample size. Since the assays were done on four consecutive days, four times more sample was required than when it was done only once. The results of the diagnostic test will now take three additional days and hence the patient will get his report three days later. The Apte procedure would also increase the cost of the test as the assays have to be carried out four times. This will result in extra expense on the part of the patient.

We believe that the unambiguity and the certainty in the results more than offset all the above drawbacks. The consulting doctor giving out the report will feel confident about the diagnosis, which is of paramount importance for the patient. With the definitive diagnosis, the management of the patient will be done in the most appropriate manner.


Acknowledgement

I am very much thankful to Prof. Maurice Mahoney of the Yale University, USA, and Dr. Dhavendrakumar of the Cardiff University, UK for their valuable suggestions. I am also thankful to Ms. Samhita Mhatre and Ms. Nilambari Pawar for helping me in the preparation of the manuscript.

References

1.
.Apte BN A novel approach to the diagnosis of enzyme deficiency, abstracted in the Bombay Hospital Journal 2000; 42: 531.
2.
. Baglioni C (1963) Correlation between genetics and chemistry of human haemoglobins.Molecular Genetics, part I (eds), Academic Press, New York, 405- 38.
3.
. Bruns GAP, Leary AC, Regina VM, Gerald PS Lysosomal Beta- -D-Galactosidase in man-hamster somatic hybrids. Cytogenet Cell Genet 1978; 22: 177-81.
4.
. Baum H, Dodgson KS, Spencer B The assay of arylsulphatase A and B in humanurine. Clin Chim Acta 1959; 4: 453-60.
5.
. Dubois G, Turpin JC, Baumann N Absence of ASA activity in healthy father of a patient with Metachromatic leukodystrophy. N Engl J Med 1975; 293:302-10.
6.
. Epstein CJ, Goldberger RF, Young DM, Anfinson CB A study of the factors influencing the rate and extent of enzymatic reaction during reoxidation of reduced ribonuclease. Arch Biochem Biophys 1962; suppl I: 223-30.
7.
. Galjaard H (1980) Genetic metabolic diseases: Early diagnosis and prenatal analysis (eds) Elsevier/North Holland Biomedical press, New York. Oxford.
8.
. Garen A, Levinthal C A fine structure genetic and chemical study of the enzyme alkaline phosphatase in E. coli. Biochim Biophys Acta 1960; 38: 470-63.
9.
. Jacob F, Monod J Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 1961;3: 318-56.
10.
. Kolodny EH, Fluharty AL (1995) Metachromatic leukodystrophy and multiple sulphatase deficiency: sulphatide lipidosis. In Scriver CR, Beaudet AL, Sly WS, Valle D (eds) in The metabolic and molecular bases of inherited disease, 7th edn, New York: McGraw-Hill, 2693-2739.
11.
. Neufeld EF Lysosomal storage diseases. Ann Rev Biochem 1991; 60: 257-80.
12.
. Porter MT, Fluharty AL, Kihara H Metachromatic leukodystrophy: arylsulphatase A deficiency in skin fibroblast cultures. Proc Natl Acad Sci,USA 1969; 62: 887-93.
13.
. Rotman B Measurement of activity of single molecules of Beta-galactosidase. Proc Natl Acad Sci, USA 1961; 47: 1981-85.
14.
. Rotman B (1970) Partial loss of activity of individual molecules of aged Beta- galactosidase. In Beckwith JR, Zipser D (eds, The lactose operon, Cold Spring Harbor Laboratory, 279-89.
15.
. Shapiro LJ, Alek KA, Kaback MM, et al Metachromatic leukodystrophy without arylsulphatase A deficiency. Ped Res 1979;13: 1179-84.
16.
. Shows TB, Scrafford-WolfL, Brown JA, Meister M Assignment of Galactosidase gene (α-gal A) to chromosome 3 in man. Cytogenet Cell Genet 1978;22:219-22.
17.
. Sips HJ, de Wit-Verbeek HA< de Wit J, Westerveld A, Galjaard H The Chromosomal localization of human Beta-Galactosidase revisited: a locus for Galactosidase on human chromosome 3 and for its protective protein on human chromosome 22. Hum Genet 1985; 69: 340-45.
18.
. Wolf B (1995) Disorders of biotin metabolism. In Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic and molecular bases of inherited disease, 7th edn,New York: McGraw-Hill, 3151-77.
19.
. Wolf B, Greer RE, Allen RJ, Goodman SI, Kien CL Biotinidase deficiency: the enzymatic defect in late onset multiple carboxylase deficiency. Clin Chim Acta 1983;131: 273-81.
20.
. Yanofsky C The tryptophan synthetase system Bact Rev 1960; 24: 221-45.

JCDR is now Monthly and more widely Indexed .
  • Emerging Sources Citation Index (Web of Science, thomsonreuters)
  • Index Copernicus ICV 2017: 134.54
  • Academic Search Complete Database
  • Directory of Open Access Journals (DOAJ)
  • EBSCOhost
  • Google Scholar
  • HINARI Access to Research in Health Programme
  • Indian Science Abstracts (ISA)
  • Journal seek Database
  • Google
  • Popline (reproductive health literature)
  • www.omnimedicalsearch.com