A Novel Approach To The Definitive Diagnosis Of EnzymopathiesCorrespondence 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 â€“ 400 014.
Tel. Clinic - 2410 5924, 2415, 2751 Res. -2515 1226
Mob. - 9821461434 Email - firstname.lastname@example.org
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.
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 220.127.116.11) 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.
Leukocyte Enzyme Estimation
Blood samples (10ml) were collected in heparinised bulbs from the patient, the patientâ€™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 patientâ€™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 18.104.22.168) (4),(11),(12) and one enzyme in serum, Biotinidase (EC 22.214.171.124), 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 â€“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 %.
Studies on one leukocyte enzyme, one urinary enzyme and one serum enzyme are presented here as illustrative examples.
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).
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 patientâ€™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).
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 â€“ 3, the patientâ€™s father FP â€“3 and mother MP â€“ 3NP â€“ 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).
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
(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 patientâ€™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 patientâ€™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
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 patientâ€™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 patientâ€™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 patientâ€™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 â€“ native and the mutant ones â€“ 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 â€“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 â€˜stabilityâ€™ 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 â€˜stabilityâ€™ 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.
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.
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