Serum malate dehydrogenase isoenzymes as indicators of severe cellular injury* INTRODUCTIOK
Malate dehydrogenase (L-malate: NAD oxidoreductase E.C. 1.1.x.37) has been shown to be localized in two cellular compartments, the cytoplasm (C-MDH) and the mitochondrion (M-MDH)1>2. Bticher et al. have demonstrated that the cytoplasmic and mitochondrial forms can be differentiated on the basis of pH optima and substrate affinityD*4. Electrophoretic separation of serum and of tissue extracts have also revealed the existence of multiple forms of MDH 5~~.However, unlike the isoenzymes of lactic dehydrogenase, the isoenzymes of MDH have not received much clinical attention. This is because the isoenzymic variants are not tissue specific. All tissues examined display two forms in relatively constant proportions 6. The elevation of the serum enzyme level or the appearance of both isoenzymes in the serum therefore does not provide diagnostic information as to their tissue origin. Indeed, because of the high cellular content, the ubiquitous tissue distribution, and the observed elevation in a multiis conplicity of pathological states 7- 10, the increase of serum malate dehydrogenase sidered, in itself, of little clinical valuell. The most that can be said is that the appearance of M-MDH in the serum is indicative of severe cell destructionr*. As part of a large scale study of serum enzymes and isoenzymes of patients in a shock-trauma unit, we have routinely determined serum malate dehydrogenase isoenzymes. The rationale for such a study was the belief that the appearance of both isoenzymes in the serum would be an indicator of severe cellular dysfunction. This communication presents the results from a series of 120 patients and indicates the utility of serum malate dehydrogenase isoenzyme determinations as an index of the extent of cellular disorganization. METHODS
Blood samples were obtained by intravenous catheter from patients in the Shock-Trauma Recovery Unit as soon as feasible after their admission. Malate dehydrogenase isoenzymes were separated by acrylamide gel electrophoresis. Seven and one half yO gels were prepared from Cyanogum [email protected]
in pH 8.6, Tris-glycine buffer (0.063 M in glycine), with 0.5% dimethylaminopropionitrile and o.I~~/” ammonium persulfate as initiator and catalyst respectively. The same buffer was used in the chambers of the electrophoresis apparatus. Fifty ,~l of serum were mixed with an equal volume of 409/~ sucrose. Five or ten ,ul of the dense mixture were carefully layed upon the upper surface of the gel in the fully assembled apparatus. Electrophoretic runs were for 60 min at 250 V. Migration was visualized with bromphenol blue tracking dye. After the electrophoretic separation, the extruded gels were incubated in the following mixture to visualize malate dehydrogenase isoby National jfGM-r,j~oo.
* Supported Grant
35 (1971) pr-501
0.1 M potassium
; 6.1 x [email protected]
1.51 mM nicotine
tetrazolium (0.5 mg/ml); 1.63 x IO-~M phenazine methosulfate (5 pg/ml). Following incubation for 2 h at room temperature in the dark, the gels were washed, fixed, and stored in 10% acetic acid. The presence or absence and the intensity of staining the M-MDH band was judged by two independent observers, who did not know the nature of the patient’s injury or his clinical status. RESULTS AND DISCUSSION In this study, 202 malate dehydrogenase isoenzyme determinations were done on the serum of 120 patients. From 76 patients only one serum sample was obtained, usually an initial one, obtained as soon as feasible after their admission to the ShockTrauma Unit. From the others, up to II additional specimens were obtained during their stay in the unit. In 24 of 44 patients from who repeated samples were obtained, the MDH isoenzyme pattern did not change. Of the 20 remaining patients, II showed a decrease in the intensity of staining of the M-MDH band; 5, an increase. In Table I, the results are presented so as to indicate the absence, a faint, or intense band of the mitochondrial isoenzyme of malate dehydrogenase. These groups are further sub-divided to indicate whether the patients survived or died. It is evident that the appearance of the mitochondrial isoenzyme in the serum provides an indication of the probability of survival. Of the patients whose serum did not exhibit the mitochondrial isoenzyme, only 14 "; died. In contrast there was 53”/0 mortality among the patients whose serum demonstrated an intense band of this isoenzyme. With few exceptions, the serum of all patients exhibited the C-MDH isoenzyme. TABLE
Patients with single determination or no change in pattern n = IO0 Patients with change in pattern (final pattern) n = 20 Totals n = 120
That the accentuated appearance of both isoenzymes of malate dehydrogenase should be seen in the serum of patients with a high risk of mortality is understandable from our knowledge of the cellular localization of the isoenzymes. The appearance and persistence in the serum of an enzyme localized in mitochondria must reflect extensive cellular necrosis or a high degree of cellular dysfunction. It follows that these pathological situations are present in patients transferred to a Shock-Trauma Recovery Unit. It may be pertinent that the 3 patients who expired and in whose serum the mitochondrial isoenzyme was absent were diagnosed as having profound septicemia. Clzn. Chin,.
l’urther, on post-mortem examination their tissues did not reveal extensive areas of cellular disruption in comparison with the tissues of other trauma patients. The patients who expired and whose serum did exhibit the mitochondrial isoenzyme were, in general, those who were admitted with extensive trauma and who were in shock. The other group of patients with an elevated M-MDH who did not survive, upon admission were not in shock or severely traumatized. However, during their stay in the unit, their status deteriorated and they exhibited clinical signs of sepsis and renal failure. Post-mortem examination of both of these groups of patients revealed areas of extensive tissue destruction. The patients with an elevated M-MDH isoenzyme pattern who survived generally fell into 3 groups: (a) severely traumatized patients in shock who were young (i.e. less than 25 years), (b) traumatized patients not in shock who responded to therapy and (c) patients admitted to the unit following extensive surgerywhose elevated serum enzymes could be related to the tissue injury accompanying the surgical procedures. From these results, it is possible to conclude that: the absence of the mitochondrial isoenzyme of malate dehydrogenase in the serum of patients is a good prognostic sign. Its presence in serum is associated with a poor prognosis especially if the patient is severely traumatized, is in shock or is elderly. This conclusion arises from the subcellular localization of the isoenzyme; its appearance in the serum being indicative of severe cellular disruption.
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