Journal Article Enzymic measurement of total bile acids adapted to the Cobas Fara centrifugal analyzer. Get access N Q Hanson, N Q Hanson Dept. of Lab. Med., Univ. of Minnesota, Minneapolis 55455-9980 Search for other works by this author on: Oxford Academic Google Scholar E F Freier E F Freier Dept. of Lab. Med., Univ. of Minnesota, Minneapolis 55455-9980 Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 35, Issue 7, 1 July 1989, Pages 1538–1539, https://doi.org/10.1093/clinchem/35.7.1538a Published: 01 July 1989

We developed an automated immunonephelometric assay for the measurement of apolipoprotein B (apo B) with a light-scattering microcentrifugal analyzer. Pretreating specimens with a dilute solution of Tween 20 or triglyceride lipase decreased the nephelometric response of apo B. Polyethylene glycol is included in the reaction mixture, and the reaction is complete within 4 min. The method is precise (CV = 6.5%, mean = 0.68 g/L) and the standard curve is linear to an apo B concentration of 2.8 g/L. Lipemia does not interfere with the method if grossly lipemic specimens are centrifuged to remove chylomicrons.

We developed an automated immunonephelometric assay for quantification of human apolipoprotein A-I (apo A-I) with a fluorescence light-scattering microcentrifugal analyzer. The presence of polyethylene glycol and Tween 20 in the reaction mixture ensures maximum exposure of the antigenic sites of the apoprotein so that immune complex formation occurs more rapidly (reaction is complete within 2 min) and to a greater extent. Lipemia and hemolysis do not interfere with the measurement of apo A-I. The method requires only 10 microL of specimen and is fast and easy to perform. Results vary linearly with apo A-I concentrations to 2.5 g/L. Assay precision (CV) was 3.1% for a specimen with an apo A-I concentration of 1.45 g/L, and the lower limit of detection was 0.15 g/L. Values for a candidate Reference Material agree well with those reported in an international survey (Clin Chem 1985;30:223-8).

Journal Article Temperature control better than calibrators for precision in the microcolumn method for hemoglobin A1c. Get access N Q Hanson, N Q Hanson Search for other works by this author on: Oxford Academic Google Scholar M L Anderson, M L Anderson Search for other works by this author on: Oxford Academic Google Scholar E F Freier E F Freier Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 31, Issue 2, 1 February 1985, Pages 339–340, https://doi.org/10.1093/clinchem/31.2.339 Published: 01 February 1985

We read with interest the recent article by Boag et al' concerning abnormally low creatinine clearance in untreated patients with anorexia nervosa. From their data the authors assumed that these patients had a reversible impairment of renal function. We, however, offer an alternative explanation for the low creatinine clearance. In normal subjects creatinine clearance calculated from the serum and 24 hour urinary creatinine measured by the Jaffe reaction closely approximates the glomerular filtration rate. This close approximation, however, is due to two compensating errors: firstly, in normal subjects 2-3 mmol (0-23-0-34 g), representing 20-30% of the urinary creatinine, is from tubular secretion rather than glomerular filtration; and secondly, 15-35 jAmoI/l (0-17-0-40 mg/100 ml), representing 15-30% of the serum creatinine as measured by conventional Jaffe methods, is from noncreatinine chromogens.2 Less than 5% of urinary creatinine measured by the Jaffe reaction is due to these non-creatinine chromogens.2 The two errors cancel each other on calculation of creatinine clearance, as one is in the numerator and the other is in the denominator of the clearance equation. In various pathological conditions the relative magnitude of these errors no longer quantitatively compensate each other, and creatinine clearance becomes a poor predictor of glomerular filtration rate. In severe renal failure creatinine clearance gives a significant overestimate of the glomerular filtration rate. Although tubular secretion still accounts for 20-30% of urinary creatinine, serum non-creatinine chromogens still account for only 1535 ,umol/l (0-17-0-40 mg/100 ml) of the serum creatinine (Jaffe reaction) and now represent .<5% of the total serum creatinine.3 In patients with low muscle mass who have low serum and 24 hour urinary creatinine creatinine clearance gives a significant underestimate of the glomerular filtration rate. In these patients the Jaffe reaction still measures urinary creatinine relatively accurately, but now the noncreatinine chromogens in the serum may represent 50% or more of the serum creatinine (measured by Jaffe reaction). A particularly dramatic case, which we encountered several years ago, was a woman weighing 45 kg with congenital anterior horn cell disease and severe wasting of the muscles. Her 24 hour urinary creatinine was only 1-2 mmoVI (136 mg) and her serum creatinine was 35 ,umol/l (0 040 mg/100 ml) by a Jaffe method, yielding a creatinine clearance of 25 m/min. Her serum urea concentration was normal at 3.9 mmol/l (23 mg/100 ml). Her creatinine clearance was artefactually low because non-creatinine chromogens accounted for >60% of her serum creatinine measured with the Jaffe reaction, and her renal function was normal. Among the principal non-creatinine chromogens that react to a Jaffe method in serum are various ketoacids.2 In patients with diabetic ketoacidosis' and a normal fasting subject3 raised acetoacetate concentrations cause spurious increases of serum creatinine measured by the Jaffe method and thus spuriously low creatinine clearances. With prolonged starvation as seen with anorexia nervosa relatively severe ketosis would be expected.6 Consequently,

We measured total serum bile acids on a fluorescence-light-scattering micro centrifugal analyzer by the direct enzymatic method with 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) and with resazurin as a fluorogenic electron acceptor. We found that serum protein has an inhibitory effect on the measurement of bile acids, but this effect was eliminated by adding bovine serum albumin to the reaction mixture in a final protein concentration (12.2 g/L) that was high compared with that contributed by a normal serum specimen. The assay is a sensitive method that reaches equilibrium in 5 min. The method is microscale (5 microL of sample, 150 microL of working reagent), is easy to perform, and is accurate (analytical recovery = 104.1%) and precise (CV = 11.1 and 5.7% on specimens with bile acid concentrations of 7.6 and 35.4 mumol/L, respectively). Normal values are 1-12 and less than 9 mumol/L on nonfasting and fasting individuals, respectively. Pure 3 alpha-hydroxysteroid dehydrogenase must be used: we found several enzyme preparations that gave falsely high values for bile acid.

Endogenous NADH-generating enzymes must be inactivated before total serum bile acids can be measured accurately by the direct enzymic method. To do this, we pretreat the sera with NaOH, in a final concentration of 0.1 mol/L. Consequently, lactate dehydrogenase activity at least as high as 30 000 U/L is destroyed, obviating blank determinations. Values for bile acid in serum, so obtained, agree with values obtained after pretreatment with heat, an alkali-methanol solution, or sodium pyruvate, but our pretreatment has the advantages of ease, speed, economy, and negligible blank values.

We observed factitious hypophosphatemia in a patient who was receiving large amounts of intravenous mannitol. Mannitol concentrations as low as 25 mmol/L inhibited phosphorus measurement by the Dupont aca endpoint method; a kinetic method was unaffected. The mechanism of the mannitol interference was binding to molybdate in the reaction, decreasing both the rate of color development and the endpoint measurement. We conclude that the molybdate concentration in the DuPont method is suboptimal for measuring phosphorus in specimens containing mannitol.

A simple, convenient, and rapid method for determining ammonia in plasma by the glutamate dehydrogenase reaction is described for the centrifugal analyzer. The measuring principle is fixed-time, with NADH as the coenzyme. ADP is added to stabilize glutamate dehydrogenase and prevent interference from endogenous plasma ADP. The reaction is linear to 400 mumol of ammonia per liter. The plasma sample volume is 100 microliter and the whole procedure takes only 25 min, including the 15-min preincubation. The normal range for venous plasma was 44 +/- 13.5 (SD) mumol of ammonia per liter.

We assessed, in 98 patients with cancer, the diagnostic value of measuring serum alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase, and glutamate dehydrogenase activities as an aid to detection of liver metastases. All four enzymes showed diagnostic value, but 5'-nucleotidase appeared to have the greatest. It showed the lowest false-positive results (7.4%) with the highest predictive value of a positive test (85.7%) and agreement (81.3%).. gamma-Glutamyltransferase showed the lowest proportion of false-negative results (2.8%), but was the least specific 35% false-positive results). Analysis of various test combinations showed that the best agreement (77.5%) was obtained when the patients were divided into those who had no or only one abnormal test result, and those who had two or more abnormal test results. However, this was not better than the agreement for 5' nucleotidase alone (81.3%). The agreement of 5'-nucleotidase and gamma-glutamyltransferase (i.e., both tests were positive or negative) was excellent (91.4%), but such agreement included only 67% of the patients with liver metastases.

Sodium, potassium, and chloride levels are artifactually depressed in hyperlipemic sera. Accurate electrolyte levels are needed for management of patients with hyperlipemia, but present methods for correcting the values (serum water and/or osmolality determinations) either are technically cumbersome or fail to provide accurate data to correct the falsely low levels. Alternatively, to determine true sodium, potassium, and chloride concentrations in hyperlipemic sera, only the required electrolyte values and the triglycerides are measured. The percentage by which the measured electrolyte levels in the hyperlipemic sample must be increased to approximate the true values is given by the following equation: per cent increase = 2.1 X triglycerides (Gm./dl.) - 0.6.

We compared two methods for serum thyroxine measurement by competitive protein binding - the Murphy and Pattee and the Seligson and Seligson methods. We found the Seligson and Seligson method, which requires less sample volume and analysis time, to be the method of choice on the basis of sensitivity, extraction efficiency, precision, and accuracy. Standard plots of time/10,000 counts versus thyroxine concentration are linear to 20 ug/dl, extraction efficiency is 99.6 per cent, within-day S.D. plus or minus 0.28 ug/dl, and 98.3 per cent of added thyroxine is recovered in the Seligson and Seligson method. The Seligson and Seligson method is superior to the Murphy and Pattee method with respect to all these parameters. The methods were also compared with correlation and normal value studies.

Kinetic methods are described for determination of total, heat-stable, and urea-stable lactate dehydrogenase (EC 1.1.1.27) activity on the centrifugal analyzer. In the urea-stable method we present, 2.6 molar urea is used to differentiate lactate dehydrogenase originating from heart and liver. This urea concentration, greater than that used by most other investigators, better differentiates between cardiac and liver lactate dehydrogenase and is as sensitive and specific for cardiac lactate dehydrogenase as the heatstable method; excellent correlation was obtained on 200 specimens assayed by both methods. The ureastable method has the advantages of speed and simplicity, better precision, and decreased serum volume, and is, therefore, preferred to the heat-stable method for determination of cardiac lactate dehydrogenase isoenzymes.