Study of certain keto-acids in metabolic disorders with particular reference to hepatocellular disease and diabetes mellitus

Abstract

The studies presented in this thesis demonstrate the diverse pathological conditions in which the keto-acids are of interest, because of their biological importance as highly active metabolic intermediates.

In addition,the inadequacy of available methods is demonstrated; as yet there is no method suitable for the separation and estimation of all known keto-acids,although a step forward has recently been made by Kaser,Kaser & Lestradet(l96o),who managed to separate KIV—hydrazone from those of KIC and KMV.The demonstration of the presence of the "branch chain" keto-acids" by he Schepper,et al in 1958,caused "an impetus to the waning interest in keto-acids"(Smith,i960). Even though methods available are imperfect,the discovery of these compounds hass"opened a new door"into the study of inborn errors of metabolism,with the description of "Maple Syrup disease", which was discussed in Part I.Mo doubt methods will be devised for the separation of the "branch chain" keto-acids which remain,as yet, unseparated,thus providing a useful means of studying the intermediary metabolism of tjie corresponding amino acids.

There seems to be no doubt that more keto-acids will be identified in material from human subjects.Thus,Biserte and Dassonville(l956) ,and McArdle(l957) have noted small traces of a hydrazone with low Rf value,but of unknown nature,and infrequent appearance,in urine. The present author has also/oted occasionally a small, intensely yellow,ultra-violet opaque spot which travels immediately in front of the a—KG hydrazone on chromatograms prepared from the extracts of concentrated urine. The quantity of the unknown was so small that attempts to elute it and examine its absorption spectrum in MaOH failed,and the present author does not know its nature,but he feels that it may be a dicarboxylic acid,because of its low Rf.

The tedious,prolonged nature of paper chromatographic methods is an additional limitation in the study of clinical problems, and the introduction of a full range of rapid,specific methods comparable to the enzymatic methods available for the estimation of a-ketoglutaric acid and pyruvic acid,would be of great help. A good deal of information is being obtained about the role of a-ketoglutarate and pyruvate in various clinical conditions, by the application of these enzymatic methods,notably by Marks(l96l) and his colleagues, but the knowledge of the'role of the "branch chain" compounds is in a most rudimentary form.

Unfortunately the clinical chemist must work with limitations not shared by his academic biochemical colleague;thus the former has only a relatively few sources of material to analyse. In the work described above in section 3.4, the aim had been to attempt to demonstrate the deamination of glutamic acid,during,or immediately after absorption,but the only material available for analysis was peripheral venous blood,the concentration of a—KG in which reflects the possible activity of the gut mucosa upon the absorbed glutamic acid,but also reflects the much greater activity of the liver.

Thus a measurement of the keto-acid content of the portal blood would have been of great help in defining the transaminase activity of the gut towards glutamic acid,much in the same way as Neame and ¥iseman(l957 &I958 ) studied the phenomenon in experimental animals. Attempts were made to study the portal vein keto-acids in blood obtained at laparotomy,but it soom became evident that this proceedure such that it was not possible to control the conditions under which the samples were collected. Further,a surgeon worried about the technical problems of his craft cannot be distracted at intervals to collect blood. In common with many other workers,the study of keto-acids in disease presented here is rather superficial,and the author feels that much more information may be obtained by undertaking a detailed study of a few cases, with a simultaneous study of the other parameters relative to the particular disease. Thus the mystery of why some patients with Cushing's Syndrome present a high fasting blood pyruvate concentrations,whilst others do not,remains a m unsolved,and perhaps an extended study on the lines presented in Section 3.1 above,with estimations related to the natural history and course of the disease,would contribute to its solution. This example,however,underlines another problem of the clinical chemist: that of obtaining a source of suitable patients,for most do not come under medical care until their disease is well developed,and many require urgent therapy because of the serious prognosis of their illness,as in this particular example(¥illiams,I96l).

From the few detailed, studies on normal subjects presented in this work,it is evident that considerable daily,and even hourly, variation in the blood concentration of the keto—acids occurs. The author feels that an extension of this type of study,both in normal subjects and patients with various diseases may reveal further interesting information,in the same way that detailed studies e revealed the loss of normal diurnal adrenal activity in (lushing's Syndrome (Forsham,I96I;Prunty,1960) This type of project,however,requires circumstances in which it is possible to supervise patients closely, and which the clinical chemist can seldom achieve.Further,it rquires patients with sufficient courage to withstand multiple venepunctures. On the basis of this and other studies undertaken by many workers,the diagnostic and prognostic value of blood concentrations of the keto-acids appears to be strictly limited,although the author feels that it will be necessary to undertake much more work,both on devising suitable methods and on the study of disease,before this impression is confirmed. The value of blood keto-acid concentrations in the past,appears to have been the way in which they have reflected changes in intermediary metabolism caused by some "biochemical lesions", (e. g. the demonstration of a raised concentration of pyruvic acid in the blood of beri-beri patients.) The recent discovery of "branch chain" keto-acids in blood has opened up a new field in which,already, a measurement of these compounds in both blood and urine has indicated the presence of a defect in the metabolism of amino acids in "Maple Syrup Disease.

Perhaps the most significant indication that much remains to be done lies in the words of De Schepper,Parmentier and Van der Saeghe(l958):that "the total 'branch chain' keto-acid concentration in the blood was found to be abnormal in certain diseases" (sic);the failure of the work described in this thesis to demonstrate a disease in which such an abnormality is found consistently suggests that work in this field of study must continue.