Survey of alpha-amylase activity and isoamylases in autopsy tissue.

We quantified total amylase and its isoenzymes in 22 different human tissues obtained at autopsy. Isoenzymes were separated by use of wheat-germ inhibition (WI) and electrophoresis on cellulose acetate (CA) and agarose (AG). Mean (+/- SD) total activity was highest in salivary glands (parotid 1710 +/- 897 U/g, submandibular 605 +/- 354 U/g), and pancreas (258 +/- 137 U/g). All other tissues contained 100- to 1000-fold less amylase. As assessed with WI, pancreas, jejunum, liver, placenta, testis, skeletal muscle, and spleen contained more than 90% pancreatic isoamylase. Salivary glands and thyroid contained more than 90% salivary isoamylase. All other tissues contained a mixture of the two isoenzymes. CA and AG often produced different results. For both CA and AG the most common pancreatic isoforms were P2 and S1. Salivary gland homogenates demonstrated a band migrating in the P3 position on CA. We conclude that both types of amylase isoenzymes can be found in tissues other than salivary gland and pancreas, but that their low total amylase concentrations diminish their clinical importance.

Three methods compared for isoamylase separation in tissue homogenates.

Using homogenates of autopsy tissue, we compared three widely available techniques for separating amylase isoenzymes: wheat-germ inhibition (WI), and electrophoresis on cellulose acetate (CA) or agarose (AG). WI separated amylase into two isoforms, CA into seven (three pancreatic and four salivary), and AG into nine (five pancreatic and four salivary). CA and WI had similar isoamylase detection limits (8-10 U/L) and similar imprecision in measuring percent S-type vs P-type isoamylase (within-run SD 1-2%), and they demonstrated a linear response to added S or P isoamylase. In contrast, the AG method had higher detection limits (10-15 U/L), greater imprecision (within-run SD 3%), and showed a nonlinear response to added S or P isomylase. We conclude that CA and WI have essentially equivalent assay attributes, superior to AG, but that CA resolves more amylase isoforms than WI.

Effect of creatine kinase-MM subtype composition on a CK-MB immunoinhibition assay.

Immunoinhibition (INH) by use of polyclonal anti-human CK-M antibody may be used to measure CK-MB in serum. Previous studies have shown that inhibiting antibodies prepared against purified muscle extracts may inhibit CK-MM by greater than 99%. Using patients' sera and muscle homogenates incubated with human serum, we studied the effect of CK-MM subtype composition on an INH assay. We found that with increasing time from the CK-releasing event, e.g., myocardial infarction, or with longer in vitro incubation, the proportion of CK-MM1 increased and the proportion of uninhibited CK-MM increased from 0.2% to 0.7-0.8%. As a consequence, CK-MB activity may be overestimated by as much as 1.6% of total CK when uncorrected INH results are used. Inhibition was maximal in samples containing 100% CK-MM3, the tissue subtype. Because of the time-dependent change in CK-MM subtypes, published results for INH from studies in which CK-MM purified from muscle was used may not be directly applicable to clinical specimens.

Regional creatine kinase, adenylate kinase, and lactate dehydrogenase in normal canine brain.

Following acute stroke, creatine kinase and other enzymes are released into the cerebrospinal fluid and blood from injured brain tissue. To determine whether regional differences in brain enzyme activity might exist and therefore affect the amount of enzyme released, we quantified the levels of creatine kinase, adenylate kinase, and lactate dehydrogenase in 12 regions of normal canine brain (n = 4). Adenylate kinase activity varied the least among regions (49 +/- 7 units/g), followed by lactate dehydrogenase activity (122 +/- 28 units/g). The pattern for both adenylate kinase and lactate dehydrogenase was higher activity in predominantly gray matter areas, lower activity in white matter, and intermediate activity in mixed regions. The distribution of creatine kinase brain isoenzyme and mitochondrial creatine kinase in canine brain was less predictable, showing wider variations among regions (isoenzyme, 462 +/- 116 units/g; mitochondrial, 42 +/- 20 units/g). Even cerebral gray matter demonstrated substantial regional variations in creatine kinase brain isoenzyme, ranging from 606 units/g in the parietal cortex to 329 units/g in the temporal cortex. We conclude that the content of creatine kinase brain isoenzyme varies more than twofold among areas of brain. This regional variation may be important in the interpretation of creatine kinase brain isoenzyme measurements in cerebrospinal fluid and serum used to assess neurologic injury following stroke.

Isoelectric focusing of neuraminidase-treated alkaline phosphatase isoenzymes on agarose gel.

We attempted to separate bone and liver alkaline phosphatase (EC 3.1.3.1) isoenzymes in human serum by isoelectric focusing on agarose gel. We found that in a pH 3-10 gradient the liver and bone isoenzymes focused into so many bands over a narrow pH range such that the information could not be quantified. However, when the bone isoenzyme in serum was first desialylated at 37 degrees C for a minimum of 6 h, catalyzed by neuraminidase (EC 3.2.1.18) at pH 5.8-6.0, we could detect four distinct bands with pls of 6.7, 6.8, 6.9, and 7.0. Under the same conditions, the liver isoenzyme in human serum focused into one band at pH 7.0. The multiple banding we observed for the desialylated bone isoenzyme has not been previously reported. The method is suited as a qualitative technique for detecting the bone alkaline phosphatase isoenzyme in serum.

Mitochondrial and MB isoenzymes of creatine kinase in cerebrospinal fluid from patients with hypoxic-ischemic brain damage.

Measurements of cerebrospinal fluid (CSF) creatine kinase (CK, EC 2.7.3.2) isoenzyme activity have been used to predict outcome in patients with acute brain injury following cardiac arrest. We identified two CK isoenzymes previously unreported in CSF from 16 patients with hypoxic-ischemic brain damage. Prior to analysis, the CK in the CSF samples was reactivated with dithiothreitol. CK isoenzymes were identified using electrophoretic and immunologic methods. Total CK activity ranged from 23 to 924 U/L (mean 452). CSF-CK-BB was the predominant isoenzyme present in all cases. In addition to CSF-CK-BB, the authors identified CSF-CK-MM in 6 cases, CSF-CK-MB in 8 cases, and CSF-mitochondrial-CK in 14 cases. The presence of CSF-CK-MM was significantly related to blood contaminating the CSF (P less than 0.02). It is proposed that CSF-CK-MB results from recombination of CK-MM and CK-BB in CSF and that mitochondrial CK is released with CK-BB into the CSF from the damaged brain tissue.

Creatine kinase isoenzymes in human cerebrospinal fluid and brain.

Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.

Cerebrospinal fluid creatine kinase activity and neurologic recovery after cardiac arrest.

We evaluated prospectively the relation between cerebrospinal fluid creatine kinase activity (CSF CK) and neurologic recovery after out-of-hospital cardiac arrest. Without knowledge of the enzyme results, we determined whether patients awoke, followed commands, or had comprehensible speech. CSF CK was significantly higher in never-awakening than in awakening patients. After cardiac arrest, elevation of CSF CK predicts poor neurologic recovery.

Cerebrospinal fluid and serum creatine kinase BB activity after out-of-hospital cardiac arrest.

In patients resuscitated from out-of-hospital cardiac arrest, neurologic outcome was compared with creatine kinase isoenzyme BB activity (CKBB) in cerebrospinal fluid (CSF) in 20 patients and in serum in 52 patients. CSF CKBB was 2 units per liter or less in patients with complete neurologic recovery but was significantly elevated in patients without neurologic recovery (mean, 55 units per liter) or with incomplete neurologic recovery (mean, 7 units per liter). Serum CKBB was detected more than 6 hours after cardiac arrest in only 4% of patients with complete neurologic but in all patients without neurologic recovery. These results demonstrate a relationship between CSF and serum CKBB and neurologic outcome after cardiac arrest.

Myoglobin concentrations and muscle-enzyme activities in serum after myocardial infarction and cardiac arrhythmia.

Sera from patients with myocardial infarction and cardiac arrhythmias were analyzed for myoglobin concentration and the activities of total creatine kinase, creatine kinase isoenzyme-2, and lactate dehydrogenase isoenzyme-1 at the time of hospital admission and during the first few days of hospitalization. The nine patients with a final diagnosis of myocardial infarction had abnormally high values for total creatine kinase, creatine kinase-2, lactate dehydrogenase-1, and myoglobin. Myoglobin concentrations were highest on admission in six patients and on the day after admission in the other three patients. Creatine kinase-2 manifested maximum activity on the day after admission for all patients with myocardial infarction. Lactate dehydrogenase-1 did not reach maximal values until the second or third day after admission. The six patients with arrhythmias did not show any significant increases in creatine kinase-2 or lactate dehydrogenase-1. Myoglobin and total creatine kinase, however, were increased in the four patients who had received cardioversion. The specificity and diagnostic usefulness of these serum measurements are discussed.

Evaluation in dogs of cross-circulation in the treatment of acute hepatic necrosis induced by yellow phosphorus.

The efficacy of cross-circulation in the treatment of acute liver failure has been evaluated in dogs. Four of 5 dogs administered a dose of yellow phosphorus that is lethal 90% of the time survived after treatment by cross-circulation of whole blood for between 1 and 8 hr with a normal dog. In 2 normal unmatched dogs plasma cross-circulations were performed over a period of 31 days without any clinical or laboratory manifestation of hypersensitivity except for lymphocytotoxic antibody titer rise. The results suggest that whole blood cross-circulation is effective and imply that a single donor could be utilized for prolonged periods of plasma cross-circulation with avoidance of immunological consequences of whole blood exchange.

Evaluation of adenosine 5'-monophosphate and fluoride as adenylate kinase inhibitors in the creatine kinase assay.

Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase.

A more sensitive automated method for determination of ornithine carbamoyltransferase activity in human serum.

Ornithine carbamoyltransferase (EC 2.1.3.3) activity is a sensitive, specific indicator of hepatocellular injury. This paper describes development of an improved automated procedure for measurement of this activity. Triethanolamine-ethylenediaminetetraacetate is used as a buffer, and activity is determined by measuring the concentration of the product, citrulline. Kinetic studies have been performed to determine optimal pH and L-ornithine and carbamoyl phosphate concentrations. Recovery of citrulline was studied. The upper limit of normal obtained in a study of 106 blood-bank donors was 6 U/liter. The automated procedure developed as a result of these studies, in which optimal assay conditions are used, produces a threefold increase in sensitivity and permits use of a sample volume of 1 ml.