Is determination of creatine kinase-2 after electrophoretic separation accurate?

Since 1991, the Ontario Laboratory Proficiency Testing Program has assessed the analytical performance of creatine kinase (CK; EC 2.7.3.2) isoenzyme-2, using fresh human serum supplemented with purified human CK isoenzymes. In Ontario, the 142 laboratories licensed to analyze CK-2 use a variety of methods: electrophoresis-based, immunoinhibition, and mass assays. During a 1992 survey, duplicate CK-2 samples with different total CK activities showed poorer precision when analyzed after electrophoretic separation than by any other method. A 1993 survey designed to validate these observations conclusively showed that electrophoresis-based assays are subject to a bias proportional to the total CK activity. These survey results were confirmed by studies with selected patients' specimens. We therefore conclude that electrophoresis-based assays may not warrant their reputation as the gold standard for CK isoenzyme measurement.

Testing for creatine kinase and creatine kinase-2 in Ontario: reference ranges and assay types.

In 1991, 246 and 136 Ontario laboratories performed total creatine kinase (CK; EC 2.7.3.2) and creatine kinase-2 (CK-2) assays, respectively. A questionnaire mailed to these laboratories requested information about the types of assay used, the origin of their reference ranges, and the source of their instruments and reagents. All laboratories used current test formulations for CK, although seven laboratories did not assay at 37 degrees C. For CK, 69% of all laboratories reported different upper reference limits for men and women (5th-95th percentiles: 160-250 and 115-215 U/L, respectively); 31% reported similar ranges for both sexes. Fifty-six percent derived their own ranges; the remainder used either kit inserts or literature references, and nearly 60% of this latter group claimed to have validated these suggested ranges before use. For 6% of all laboratories, their pediatric ranges were similar to their adult ranges. For CK-2, only 32% used their own reference range; the remainder used kit inserts or literature references, but only 49% of this group validated these ranges before use. Reference limits (5th-95th percentiles) for CK-2 were as follows: activity 6-24 U/L; fraction of CK, 0.022-0.06; and, for mass assays, 5-10 micrograms/L and relative index 0.015-0.04.

Results of a province-wide quality assurance program assessing the accuracy of cholesterol, triglycerides, and high-density lipoprotein cholesterol measurements and calculated low-density lipoprotein cholesterol in Ontario, using fresh human serum.

To evaluate laboratory performance, eight to 13 samples of fresh human serum from volunteers were sent to 250 laboratories in the Canadian province of Ontario licensed to perform lipid analysis. Fresh human specimens were used because of potential matrix effects with processed materials. We show that on all survey samples, 71% (range, 63% to 82%) of participating laboratories are within +/- 5% of the target cholesterol value and that 93% are within +/- 10%. The goal of the National Cholesterol Education Program for 1992 is total error of no more than +/- 9% for 95% of results. The unblanked triglycerides results show that on all samples 40% (14% to 59%) of participants are within +/- 5% and 68% (range, 31% to 86%) are within +/- 10% of the target value. For triglycerides results from 0.9 to 2.0 mmol/L, 80% or more are within +/- 0.2 mmol/L. Between 2.0 and 3.0 mmol/L, 90% are within +/- 0.3 mmol/L of the target values. For high-density lipoprotein cholesterol, for all samples 35% (range, 24% to 50%) of laboratories are within +/- 5% and 68% (range, 55% to 88%) are within +/- 10%. A range of 80% to 95% of participants are within +/- 0.2 mmol/L of the target values. For calculated low-density lipoprotein cholesterol, 51% and 62% of the laboratories surveyed are within +/- 5%, with 83% and 89% within +/- 10% of the target values. We conclude that the laboratory measurement of lipids is approaching the degree of accuracy and precision required for clinical purposes, and that the use of fresh human serum samples is a viable approach to their proficiency testing.

Proficiency testing for creatine kinase isoenzyme CK-2 (CK-MB) in Ontario.

Three surveys of the measurement and interpretation of creatine kinase (CK; EC 2.7.3.2) isoenzyme 2 (CK-MB) were conducted in Ontario, Canada, in 1989. Of the clinical laboratories participating, 66% used immunological methods and 24% used electrophoretic methods. Although reference ranges and interpretative routines varied widely, 95% of the laboratories reported correct interpretations for 10 of the 15 vials tested. The only major problems occurred with samples with very low total CK activity. Within-survey duplicate results compared well, and 89% of the laboratories had consistent between-survey results, even for specimens with low total CK activity. Errors were proportional to the frequency of use of the different analytical methods. The lyophilized testing material gave higher results with methods for measuring the mass of CK-2, suggesting that the material contained inactive but immunologically intact CK-2. The surveys indicate that laboratories should review their protocols for measuring CK-2 when only a single sample from the patient is available.

Low-glycemic-index starchy foods in the diabetic diet.

Eight patients with noninsulin-dependent diabetes underwent two 2-wk study periods in random order during which they were provided with carbohydrate foods with either a high or low glycemic index (GI). Over both high-GI and low-GI periods there were significant reductions in body weight, serum fructosamine, and cholesterol. Reductions in fasting blood glucose, HbA1c, and urinary c-peptide-to-creatinine ratio were significant only over the low-GI period despite a smaller mean weight loss. Reductions in triglyceride were significant only over the high-GI diet. Inclusion of low-GI foods into diets of patients with diabetes may be an additional measure that favorably influences carbohydrate metabolism without increasing insulin demand.

Associations of plasma high-density lipoprotein cholesterol with clinical chemistry data.

We examined data from the participants in the Lipid Research Clinics Program Prevalence Study for associations between high-density lipoprotein (HDL) cholesterol and clinical chemistry tests. There is a negative relationship between serum thyroxine and HDL cholesterol: men 20-69 years and women 20-44 years with low thyroxine levels have significantly higher HDL cholesterol than those with high thyroxine levels. Women 45-69 years with hyperglobulinemia (> 3.2 g/dl) have significantly lower HDL cholesterol levels than those with lower globulin levels. There is a weak negative association between HDL cholesterol and serum uric acid in men 20-44 years and a stronger association in women. A weak negative association between HDL cholesterol and plasma glucose is present only in men 20-44 years and women 45-69 years. Subjects with high serum bilirubin or serum aspartate aminotransferase (AST, SGOT) values have higher HDL cholesterol levels that are statistically significant in men 20-69 years and women 20-44 years (bilirubin) and men 45-69 years and women 45-69 years (AST). There is a negative association between alkaline phosphatase and HDL cholesterol in women and young men. These results suggest that thyroid hormones may be involved in the regulation of HDL cholesterol, and that there are associations between HDL cholesterol and the clinical chemistry tests that are not necessarily explained by disease. However, in the whole population, the plasma constituents measured as clinical chemistry tests, or the mechanisms that regulate their levels, are not important determinants of plasma HDL cholesterol levels.

Pathogenesis of acidosis in hereditary fructose intolerance.

An 18-yr-old man with a classical history of hereditary fructose intolerance (HFI) developed typical biochemical changes following an oral fructose load: fructosemia, hypoglycemia, hypophosphatemia, hyperuricemia, and metabolic acidosis. Hypokalemia (3.1 meq/liter) was also noted. Three aspects of this case expand the published literature on this syndrome: (1) Metabolic acidosis was found to be due to both lactic acidosis and proximal renal tubular acidosis (RTA). We could quantitate the relative contribution of each, and found that urinary bicarbonate loss due to proximal RTA accounted for less than 10% of the fall in serum bicarbonate. The major cause of the metabolic acidosis was lactic acidosis. (2) Hypokalemia was found to be due to movement of potassium out of the extracellular space rather than to urinary loss. Potassium may have entered cells with phosphate or may have been sequestered in the gastrointestinal tract. (3) The coexistence of proximal RTA and acidemia made it possible to study the effect of acidemia on the urine-blood partial pressure of carbon dioxide (PCO2) gradient in alkaline urine (U-B PCO2). The U-B PCO2 measured during acidemia was much higher at the same urine bicarbonate concentration than in normal controls during alkalemia, providing evidence in humans that acidemia stimulates distal nephron hydrogen-ion secretion.

The mechanism of heparin stimulation of rat adipocyte lipoprotein lipase.

Free fat cells and stromal-vascular cells were prepared from rat adipose tissue by incubation with collagenase. NH(4)OH-NH(4)Cl extracts of acetone-ether powders prepared from fat cells contained lipoprotein lipase activity but extracts of stromal-vascular cells did not. Intact fat cells released lipoprotein lipase activity into incubation medium, but intact stromal-vascular cells did not. The lipoprotein lipase activity of the medium was increased when fat cells were incubated with heparin, and this was accompanied by a corresponding decrease in the activity of subsequently prepared fat cell extracts. Heparin did not release lipoprotein lipase activity from stromal-vascular cells. The lipoprotein lipase activity of NH(4)OH-NH(4)Cl extracts of fat cell acetone powders is increased by the presence of heparin during the assay. This increase is not due to preservation of enzyme activity, but to increased binding of lipoprotein lipase to chylomicrons. Protamine sulfate and sodium chloride have little effect on the binding of lipoprotein lipase to chylomicrons, but they inhibit enzyme activity after binding to substrate has occurred. These inhibitors do, however, inhibit the stimulatory effect of heparin on enzyme-substrate binding.