Theoretical aspects of enzymes in diagnosis. Why do serum enzymes change in hepatic, myocardial, and other diseases?

The pathophysiological mechanisms of leakage, synthesis, and decreased removal of enzyme from blood all play a role in explaining why serum enzymes increase in disease. This presentation has focused on increased synthesis as an important and perhaps major source of increased serum enzymes. The second key point has to do with the reference standard. There is tremendous sophistication in the field addressed to the numerator, that is, biochemical units. The natural tendency is to focus attention on what we want to know in the numerator, the enzyme changes in the tissue. In so doing, however, we tend to ignore the changes of the reference standard in the denominator, which influence the final result as much as the numerator. The following recommendations are made: uniformity, enzyme activities referable to morphological units, and basic biological significance. Total organ per gram initial body weight is recommended as the optimal reference standard whenever possible. If this were done, perhaps we could then resolve the dilemma of the poor understanding of why serum enzymes change in disease. We would, thereby, be better equipped to bring the major part of biochemical pathology out of its infancy as described by Farber.

Enhanced cardiac enzyme profile.

A protocol for an enhanced Cardiac Enzyme Profile is proposed based on an admission, or initial, serum specimen and a second specimen 16 hours after onset of symptoms as minimal baseline serum samples in order to accomplish several simultaneous goals: 1. Detecting CK2MB at its average peak for maximal assurance of diagnosis when release is small and for prognosis in all cases of increased serum CK2MB 2. Detection of laboratory evidence of myocardial injury when admission is delayed after onset by the collection of an admission sample for declining CK2MB, and for assays of other enzymes with longer time curves after myocardial injury such as LD isoenzymes and ASAT/ALAT activities and ratio 3. Establishment of decision limits and criteria for the determination of laboratory evidence of myocardial injury 4. Providing cost-effective procedures other than limitation of the number of samples; these include establishing thresholds and criteria for total CK, total LD, and ASAT so that isoenzymes and ALAT are only performed when thresholds are exceeded and criteria are met; performing only CK and, if the threshold is exceeded, CK isoenzymes on the 16-hour sample; collecting additional samples after the first two only when indicated by positive or suspicious (borderline) results and only on routine morning or afternoon rounds rather than specifically timed specimens (except in cases involving thrombolytic therapy); and termination of the protocol once peak positive CK2MB activity and requisite diagnostic consensus confirmation (such as positive LD isoenzymes) is obtained whether or not thrombolytic therapy is involved. Tissue localization of the enzymes has been outlined in some detail with particular reference to the amount of CK2MB in skeletal muscle. Pathophysiological factors discussed in more depth in a previous article have been amplified here with particular reference to the role of increased synthesis as a response to myocardial injury by surrounding prehypertrophic and hypertrophic myocardium as a possible major source of increased serum enzymes in myocardial infarction. ASAT and especially the ASAT/ALAT ratio are useful tests in the protocol, particularly in cases tested late after onset of symptoms when CK2MB has declined into the borderline or usual range, and ASAT/ALAT may be helpful in evaluating LD isoenzyme results. Codes for interpretive comments are provided to serve as guidelines.

Correlation of mechanical properties of vertebral trabecular bone with equivalent mineral density as measured by computed tomography.

We tried to determine whether mineral-equivalent measurements that were obtained using computed tomography could be used to predict the mechanical properties of vertebral trabecular bone. Vertebral bodies that had been obtained during routine autopsy were evaluated by computed tomography. The mechanical properties of the vertebral trabecular bone were determined by subjecting cylindrical specimens to simple compression until failure occurred. The ultimate strength and elastic modulus were determined from load time curves, using constant displacement rate loading. Atomic absorption spectrophotometry was used to determine the weight per cent calcium of each specimen, and quantitative light microscopy was used to determine area fraction bone. Significant positive correlations were found between the observed mechanical properties of the trabecular bone and the equivalent mineral density as measured by computed tomography. Compressive strength (r = 0.720), elastic modulus (r = 0.574), trabecular calcium density (r = 0.780), and area fraction bone (r = 0.579) were all correlated with the equivalent mineral density.

Liver aspartate aminotransferase activity as a power function of body weight.

Total liver enzyme activity (E) for aspartate aminotransferase (ASAT; EC 2.6.1.1) per gram body weight (W) decreases in six species as body weight increases from mice to cattle according to the equation E/W 0.85 = c, where c is a constant. The 0.85 power of body weight thereby provides a reference standard for the direct arithmetic comparison of liver ASAT activities in six species with different body weights. The use of W 0.85 should provide a tool in clinical studies in which data from experimental animals must be related to humans, e.g., in establishing the relationship between drug dosage (or toxicity) and body weight.

Effect of dietary copper on colonic tumor production and aortic integrity in the rat.

Gastrointestinal malignancy has been associated with aortic aneurysmal disease in humans, while metabolic derangement of copper has been incriminated as a possible promotor of aneurysmal development of the aorta. An animal model utilizing the carcinogen 1,2-dimethylhydrazine (DMH) was selected to evaluate levels of dietary copper on both colonic tumor production and morphologic changes in the rat aorta. Six groups, each including 10 Sprague-Dawley rats, received 16 weekly doses (20 mg/kg) of DMH beginning at 4 weeks of age. Groups were maintained on either normal (25 ppm), low (0.6 ppm), or high (100 ppm) copper chow during the entire experimental period. After 25 weeks, all animals were sacrificed to assess colonic tumor production and to perform scanning (SEM) and transmission electron microscopic (TEM) studies of the rat aorta. Results showed a significant increase in colonic tumor production (3.14 +/- 0.39 tumors per centimeter colon) in rats treated with low-copper chow and DMH when compared with rats on normal chow and DMH (0.74 +/- 0.07 tumors per centimeter colon) and animals maintained on high-copper diets and DMH (0.76 +/- 0.08 tumors per centimeter colon). In addition, morphologic study showed disruption of the intima and media in rats maintained on low-copper diet alone, and also on low-copper diet plus DMH. The results of this study showed that DMH and low dietary copper significantly increase (P less than 0.001) the yield of colonic tumors and produce loss of aortic integrity when studied morphologically. Copper levels may be important in the association of neoplasia and aneurysm formation in the clinical setting.

Source of increased serum aspartate and alanine aminotransferase: cycloheximide effect on carbon tetrachloride hepatotoxicity.

Repeated doses of the protein synthesis inhibitor cycloheximide prevented the increases in rat liver mitochondrial and cytosolic aspartate aminotransferase, in alanine aminotransferase and in protein content observed 24 h after a single carbon tetrachloride injection. Serum aminotransferase activity increases induced by carbon tetrachloride were also decreased as much as 75.7% with cycloheximide. Increased synthesis is, therefore, suggested as an important and sometimes major source of increased serum aminotransferases in hepatocellular injury. This effect of cycloheximide lends support to the hypothesis that the liver enzyme increases after CCl4 are probably due to increased synthesis, in addition to the classically held mechanisms of leakage from necrotic or damaged hepatocytes. This explanation of the mechanisms of release of aminotransferases in rat liver injury would clarify many clinical observations if the same phenomenon were to occur in humans in response to hepatic injury. These data suggest that increased serum aminotransferase activities represent a healing, in addition to a degenerative, process.

Effect of cycloheximide on increased aspartate aminotransferase in carbon tetrachloride hepatotoxicity.

The previously reported increases in liver and serum aspartate aminotransferase (ASAT) activities and liver protein content 24 hours after the administration of carbon tetrachloride (CCl4) were reduced by administering multiple doses of the protein synthesis inhibitor cycloheximide (CH). Liver ASAT and protein content were reduced to saline-injected control levels, and the serum ASAT increase was reduced by 45.0 percent in rats given CH. Although there are morphological features of severe hepatotoxicity in the cycloheximide-carbon tetrachloride-injected rats, cycloheximide does reduce the severity of these lesions and the regenerative response. These findings lend some support to the hypotheses that (1) the increase in liver ASAT activity and protein content after CCl4 is due to increased synthesis and (2) the increase in serum ASAT after CCl4 is most likely due to a combination of increased synthesis and leakage from necrotic and damaged cells.

Response of rat liver aspartate aminotransferase to carbon tetrachloride.

Rat liver aspartate aminotransferase activity per total liver per gram initial body weight was increased 24 hours after carbon tetrachloride injection suggesting that increased synthesis may be a source of increased serum enzyme activity as a response to hepatocellular injury. Protein content per total liver per gram initial body weight was also increased so that the specific activity of the enzyme was unchanged. Enzyme activity per gram liver wet weight was decreased consistent with hepatomegaly, edema, and dilution of enzyme. Total liver per gram initial body weight is suggested as the optimal reference standard.

Increased rat liver homogenate, mitochondrial, and cytosolic aspartate aminotransferase activity in acute carbon tetrachloride poisoning.

Rat liver homogenate, subcellular fractions, and sera were assayed for aspartate aminotransferase 24 h after the administration of carbon tetrachloride. Enzyme activity per total liver per gram initial body weight was significantly increased in the homogenate, and in the mitochondrial and cytosolic fractions. Protein content per total liver per gram initial body weight was also increased so that the specific activity of the enzyme was unchanged. Enzyme activity per gram liver wet weight was decreased consistent with hepatomegaly, edema, and dilution of enzyme. Enzyme and isoenzyme activities of serum were increased. These findings support the hypothesis that increased synthesis may be a source of increased serum aspartate aminotransferase and its isoenzymes as a response to hepatocellular injury.

Serum chloride analysis, bromide detection, and the diagnosis of bromism.

Current methods for determining serum chloride concentration vary in specificity. Laboratory detection of bromide in serum is usually dependent on spuriously high chloride values. In the absence of historical information, the chemical identification of bromide in serum may be the only diagnostic sign of bromism. Bromide may be found in as many as 1% of sera obtained from inpatients of a general hospital. Ion-selective electrodes and some thiocyanate methods for chloride analysis can be highly sensitive to bromide (interference). Chloride electrodes are also sensitive to iodide.