Enzyme immunoassay of urinary beta-hexosaminidase isoenzymes in patients with renal transplants

beta-Hexosaminidase (NAG) and percent of NAG B were studied in twenty patients following renal transplantation. Median urinary NAG for twenty reference individuals was 0.26 U/mmol creatinine and NAG B was 24%. Urinary NAG decreased rapidly from a median of 3.7 U/mmol on the third day, to 1.2 U/mmol on the 15th day after transplantation in the patients with no major complications. The percentage of NAG B did not change significantly during this period and did not differ from the reference population. Rejection and cyclosporine toxicity were diagnosed on 17 occasions. Urinary NAG rose more than twofold in 15 of these episodes. The percentage of NAG B was slightly increased in 6 of these. Six months after discharge 17 of the renal transplants functioned well. They exhibited a marked decrease (almost normalized) of urinary NAG with no change in the percentage of NAG B.     

Rat plasma clearance rate and organ distribution of beta-hexosaminidase isoenzymes from human serum.

beta-Hexosaminidase (EC 3.2.1.30) is markedly increased in human serum in liver disease, chronic alcoholism, and pregnancy. Knowledge of the clearance rate of plasma/serum beta-hexosaminidase is necessary to evaluate this increase. We studied the plasma clearance of beta-hexosaminidase isoenzymes (purified from human serum and placenta) after their infusion into rat circulation. A recently developed enzyme immunoassay method was used to measure the human beta-hexosaminidase isoenzymes; this method relies on both immunoreactivity and enzyme activity, so intact human isoenzymes were measured. In comparison with the placental isoenzymes (t1/2 less than 2 min), the serum forms showed a considerably slower clearance (t1/2 = 2-4 h). However, desialylation of the serum forms resulted in their rapid clearance (t1/2 less than 2 min). The organ localization of the enzyme eliminated from the circulation was investigated 24 h after infusion. Placental and native serum isoenzymes accumulated mainly in the liver and the spleen. Desialylated serum forms were almost exclusively localized to the liver.     

Enzyme immunoassay of beta-hexosaminidase isoenzymes using monoclonal antibodies

Purified beta-hexosaminidase A (Hex A) and beta-hexosaminidase B (Hex B) were used as immunogens in mice, with the purpose of obtaining isoenzyme-specific monoclonal antibodies (mabs). A total of 60 mabs were developed, 23 specific for Hex A and 37 recognizing both isoenzymes. At low pH, two of the latter mabs reacted only with Hex B, and it was, therefore, possible to develop enzyme immunoassays for the specific determination of Hex A and Hex B. The precision of the methods was adequate with intra- and interassay coefficients of variation below 3%.     

Significance of isolated increases in total lactate dehydrogenase and its isoenzymes in serum of patients with bacterial pneumonia

Total lactate dehydrogenase (LD, EC 1.1.1.27) activity in serum and LD isoenzymes were quantified at the time of diagnosis in 320 patients with bacterial pneumonia. In eighty, LD activity was increased, but this was accompanied by either other pathological results for liver-function tests or associated diseases that could explain it. The remaining 240 patients were divided into four groups, based on their total serum LD values: group A, less than 225 U/L (normal limit); group B, 226-350 U/L; group C, 351-499 U/L; and group D, greater than 500 U/L. Total LD was above normal at diagnosis in 40% of the patients. Recovery time was twice as long in group D as in groups A, B, and C. In five patients from group D, the pneumonia reflected underlying lung cancer. In groups B and C, the LD-3 ratio was increased in comparison with group A; in group D, LD-4 and LD-5 were increased up to twice the normal limit. Evidently nearly half of patients with bacterial pneumonia may show isolated increases in total LD activity (mostly LD-3) in serum. In cases with high activity, prolonged recovery time is expected. Intensive follow-up and extensive investigation are warranted in these patients, because some may have underlying lung cancer.     

N-acetyl-beta-glucosaminidase isoenzymes in serum and urine of patients with diabetes mellitus

The isoenzyme pattern of N-acetyl-beta-glucosaminidase (NAG) in serum and urine was studied in two groups of patients with diabetes mellitus and in 30 control subjects. Total NAG activity was significantly (P less than 0.001) increased in the serum and urine of the 20 diabetics with vascular complications, but was insignificantly increased in the 20 diabetics without vascular complications. Ion-exchange chromatography demonstrated the presence of two major isoenzymes of NAG, A and B. The proportion of isoenzyme A activity always exceeded that of isoenzyme B. The proportion of isoenzyme B in serum of diabetics was lower than in controls; the reverse was true for urine of diabetics. The NAG isoenzymes pattern may provide additional diagnostic information regarding diabetic status and complications of diabetes.     

Differential diagnosis of patients with abnormal serum creatine kinase isoenzymes

For the diagnosis of myocardial injury, particularly AMI, CK-MB has become the gold standard. Changing CK-MB activities in serially collected blood from patients with suggestive signs and symptoms of AMI is almost pathognomonic for infarction. Nevertheless, an increased CK-MB cannot be equated with AMI owing to the many other types of inflammatory, traumatic, and miscellaneous forms of injury to the heart and the trace activities of CK-MB in skeletal muscle. Other enzyme tests for AMI are less efficient. In order of decreasing efficiency, the tests are CK-MB, CK, LD1 greater than LD2 or LD1/LD2 greater than 0.76, AST and LD; the latter two tests are not cost effective and add little or nothing when results for CK-MB, CK, and LD isoenzymes are available. The value of the isoforms of CK-MM and CK-MB remains to be established. Early evidence suggests that they could be helpful in the diagnosis of AMI; however, owing to the greater technical difficulties in performing these tests, their use is necessarily more restricted. Enzyme testing on admission and then every 12 hours for 2 days is sufficient and effective in making the initial diagnosis. In patients presenting early after an attack, CK and CK-MB are often normal. Decisions on AMI cannot be made on blood tests collected in the emergency department. Clot-lysing agents like streptokinase, urokinase, and tPA have changed the therapy of AMI dramatically. Enzyme tests clearly separate patients with and without successful therapeutic or spontaneous reperfusion. With successful reperfusion, the uniform finding has been a "washout" phenomenon with significantly earlier peaking times for CK and CK-MB. The isoforms of CK and myoglobin give the earliest peaks after successful reperfusion. With faster turnaround times for these tests, they may become important tools in patient management.     

Biological variance of total lactate dehydrogenase and its isoenzymes in human serum

We measured the variance components of total lactate dehydrogenase (LD; EC 1.1.1.27) activity and isoenzymes in sera of 24 healthy laboratory staff, ages 23-50 years, over a 12-month period. We used the first six weeks' results to establish baseline values for each analyte in each individual. These values were "normally" distributed for total LD and isoenzymes 1 through 4, but values for isoenzyme-5 were skewed slightly to the left. The overall means and variances determined for the remaining 10 months were not significantly (p greater than 0.05) different from the corresponding baseline values. Average variances (SD2) for the longer-term (10-month) study were: intra-individual, 732, 1.92, 2.25, 1.09, 1.11, and 2.25, and inter-individual, 1988, 5.03, 1.76, 1.57, 1.21, and 2.01 for total LD (U/L) and LD-1 through LD-5 (% of total), respectively. Average long-term analytical variability was less than 35% of the total variance for the five isoenzymes and 6% for total LD, and was characteristic of the individual. There were no significant differences in mean activities with respect to age or sex. All six analytes exhibited slight seasonal variations.     

Activity of serum aspartate aminotransferase isoenzymes in patients with acute myocardial infarction

Activities of aspartate aminotransferase (AST) isoenzymes were determined in serial serum samples from 40 cases of acute myocardial infarction, and compared with activities of creatine kinase, CK-MB isoenzyme, lactate dehydrogenase, and alpha-hydroxybutyrate dehydrogenase for temporal changes. Cytosolic (soluble) AST (s-AST) and mitochondrial AST (m-AST) respectively increased 6.6 and 9.0 h after onset of chest pain. The median time at which serum m-AST activity peaked (15.8 U/L, range 6.4-53.5 U/L) was 47.8 h after the onset of infarction, 19.8 h later than the peak s-AST activity (171 U/L, range 53-517 U/L) and m-AST also disappeared from the serum more slowly than s-AST (p less than 0.001). Serum m-AST values were above normal for at least six days after the infarct. The ratio of m-AST to total AST in serum increased after myocardial infarction, being greatest (20%, range 11-32%) on the third day after onset. For individuals, peak activities of s-AST correlated well with total CK (r = 0.91) and CK-MB (r = 0.86) peak activities, indicating that s-AST also reflects the infarct size. However, m-AST correlated poorly with the enzymes commonly used in infarct diagnosis; it apparently provides different biological information.     

Methods compared for determining total amylase activity and amylase isoenzymes in serum

We evaluated two kinetic methods for determining total amylase activity and isoenzyme composition in serum. Stability studies of reagents for measuring total activity indicate that reagents containing 4-nitrophenyl-alpha-glucosides or enzyme-linked reagents can be stored only for seven days at 4 degrees C. Methods based on 4-nitrophenyl-alpha-glucoside substrates cannot be used if the reagent absorbance at 405 nm exceeds 2. However, in the alpha-amylase EPS method (Boehringer Mannheim) an ethylidene-protected 4-nitrophenyl-alpha-D-maltoheptaoside substrate is stable for up to 28 days after reconstitution. Further studies indicated that the Amylase-DS (Beckman) and the alpha-Amylase EPS standard curves are linear to at least six times the upper limit of the reference interval. Within-batch imprecision (CV less than 1.1%) and between-batch imprecision (CV less than 3.3%) for these two methods are comparable with those for other kinetic methods, and there is excellent correlation (r2 = 0.983) between the two methods. The reference interval, determined by use of samples from 90 healthy blood donors, is 31 to 141 U/L for the amylase-DS method, 22 to 92 U/L for the alpha-Amylase EPS method. We also used these two methods to measure amylase isoenzymes after inhibiting the salivary isoenzyme with either a lectin or a monoclonal antibody. We found the monoclonal antibody method more specific than the lectin inhibition method for determining the isoenzymes.     

Elevated urinary beta-hexosaminidase in patients with stroke.

Urinary beta-hexosaminidase is a sensitive indicator of renal damage. The urinary excretion of this enzyme was measured in 31 patients with ischaemic stroke in the acute phase and in 126 patients 21-43 weeks after their stroke. Both immediately and after 21-43 weeks the patients showed a similar and a significantly increased level of urinary beta-hexosaminidase. This indicates the presence of renal injury in the stroke patients, which in turn might reflect a generalized vascular disease.     

Lactate dehydrogenase and its isoenzymes in serum from patients with multiple myeloma

Concentrations of total lactate dehydrogenase (LDH; EC 1.1.1.27) and LDH isoenzyme patterns were studied in serum of 19 patients with multiple myeloma and in 19 healthy controls. Patients were divided into three groups (pretreatment, nonresponders, and responders to treatment), based on their clinical status at the time of blood sampling for LDH. The LDH values were found to be significantly higher (P less than 0.05) in the pretreatment group and in the nonresponders than in the responders and the control group, the mean +/- SE values being 445 +/- 35 and 532 +/- 75 units/mL vs 349 +/- 75 and 190 +/- 7.1 units/mL, respectively. Compared with responders and healthy controls, newly diagnosed patients and nonresponders had slight diminutions in LDH-1 and LDH-2, but increased LDH-3. We conclude that determination of LDH and its isoenzymes in serum can be of value as prognostic factors in patients with multiple myeloma.     

Diagnosis of patients with raised serum calcium level in primary care, Sweden

OBJECTIVE: To study the diagnosis of hypercalcaemic patients and to evaluate whether frequent analyses of serum calcium can detect more patients with hypercalcaemia. DESIGN: Retrospective study of serum calcium analyses performed during the time period 1992-2000 and of the medical records of patients with elevated serum calcium levels between 1995 and 2000. SETTING: Primary care in Tibro, Sweden. SUBJECTS: Patients from the local community attending the primary healthcare centre. MAIN OUTCOME MEASURES: Frequency of serum calcium analyses, hypercalcaemic patients, and their diagnosis. RESULTS: Doubling the number of serum calcium analyses did not increase the detected number of raised calcium levels. On the other hand, more frequent parathyroid hormone (PTH) analyses resulted in a corresponding increase in detected high PTH levels. In Tibro, 15% (n = 22) of the patients with hypercalcaemia were diagnosed with primary hyperparathyroidism, giving a rate of 0.22%. This is comparable to the prevalence in other population studies. Over 40% (n = 9) of patients with primary hyperparathyroidism in the study had only slightly raised serum calcium levels (2.55-2.60 mmol/l). In 70% (n = 99) of the cases, the cause of hypercalcaemia was unknown. The second most common diagnosis was skeletal disorders followed by kidney disease. CONCLUSION: An increase in the number of serum calcium analyses did not result in increased detection of raised calcium levels. In contrast, an increase in the number of PTH analyses resulted in increased detection of primary hyperparathyroidism. Therefore, PTH analyses should be used more frequently.     

Diagnosis of patients with raised serum calcium level in primary care,Sweden

Objective. To study the diagnosis of hypercalcaemic patients and to evaluate whether frequent analyses of serum calcium can detect more patients with hypercalcaemia. Design. Retrospective study of serum calcium analyses performed during the time period 1992–2000 and of the medical records of patients with elevated serum calcium levels between 1995 and 2000. Setting. Primary care in Tibro, Sweden. Subjects. Patients from the local community attending the primary healthcare centre. Main outcome measures. Frequency of serum calcium analyses, hypercalcaemic patients, and their diagnosis. Results. Doubling the number of serum calcium analyses did not increase the detected number of raised calcium levels. On the other hand, more frequent parathyroid hormone (PTH) analyses resulted in a corresponding increase in detected high PTH levels. In Tibro, 15% (n?=?22) of the patients with hypercalcaemia were diagnosed with primary hyperparathyroidism, giving a rate of 0.22%. This is comparable to the prevalence in other population studies. Over 40% (n?=?9) of patients with primary hyperparathyroidism in the study had only slightly raised serum calcium levels (2.55–2.60 mmol/l). In 70% (n?=?99) of the cases, the cause of hypercalcaemia was unknown. The second most common diagnosis was skeletal disorders followed by kidney disease. Conclusion. An increase in the number of serum calcium analyses did not result in increased detection of raised calcium levels. In contrast, an increase in the number of PTH analyses resulted in increased detection of primary hyperparathyroidism. Therefore, PTH analyses should be used more frequently.     

Total lactate dehydrogenase and its isoenzymes in serum of patients with non-small-cell lung cancer

Values for total lactate dehydrogenase (LD, EC 1.1.1.27) activity in serum and LD isoenzymes were determined at diagnosis in 273 patients with non-small-cell lung cancer, 85 of whom were in stage 1, 92 in stage 2, and 96 in stage 3. We divided the patients into three groups, based on their total serum LD values: less than 225 U/L (normal reference range), 226-500 U/L, and greater than 500 U/L. Overall values for LD were above normal at diagnosis for 69% of the patients, being moderately increased in 63 patients and highly increased in 125. Eighty percent of the patients in stage 1 had normal values for LD at diagnosis, but 88% of the patients in stage 2 and 94% of the patients in stage 3 had above-normal LD values at diagnosis. In 55% of the patients in stage 2 and 73% of the patients in stage 3, LD activity was highly increased. In the patients with normal values for total LD, the proportions of the LD isoenzymes were normal. In the patients with increased LD, the isoenzyme proportions were increased for LD-4 and LD-5, up to twice the normal values. The sensitivity of LD in detection of lung cancer was 60% for LD at the cutoff point of 250 U/L in comparison with normal controls, and 47% for LD at the cutoff point of 310 U/L in comparison with the benign lung disease group of patients (95% specificity). We conclude that total LD in serum may be a direct indicator of clinical stage and tumor burden in patients with non-small-cell lung cancer.     

beta-hexosaminidase, alpha-D-mannosidase, and beta-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.     

Abnormal alkaline phosphatase isoenzymes detected in the serum of elderly patients

Alkaline phosphatase (ALP) isoenzyme analysis of 101,832 serum samples was performed by electrophoresis using cellulose acetate membrane, and abnormal bands at the alpha(1) and alpha(2) globulin positions were detected in 23 samples. The physicochemical properties of these abnormal ALP isoenzyme fractions were examined. In brief, the abnormal fractions were heat-sensitive and inhibited by L-phenylalanine, and neither sialic acid in their polysaccharide chains nor the glycosyl-phosphatidylinositol anchor was detected by the enzyme treatment. The physicochemical properties of abnormal ALP isoenzyme fractions detected in Patients 1 - 22 were similar to those of the adult small intestine type. However, the molecular weight of the adult small intestine type abnormal fractions was smaller than that of the normal fractions. These adult small intestine type abnormal bands at the alpha(1)- to alpha(2)-globulin positions, which were identified in the serum of Patients 1 - 22, were detected in elderly patients. Most of them had various basal diseases such as renal insufficiency, fracture, interstitial pneumonia, and chronic pancreatitis. Some of them had severe diseases such as rectal cancer, descending colon cancer, and septic shock. In Patient 23, the polysaccharide chain had sialic acid and was heat sensitive physicochemical properties that were similar to those of the Kasahara ALP variant.