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.     

Assay of ornithine carbamoyltransferase activity in human liver using carbon-labeled ornithine and thin-layer chromatography

Ornithine carbamoyltransferase (EC 2.1.3.3) activity in human liver homogenates has been measured using 14C-labeled ornithine and unlabeled carbamoyl phosphate. A thin-layer chromatographic (TLC) procedure is used to separate the radioactive substrate and product, ornithine and citrulline, respectively, and the regions of the chromatogram corresponding to ornithine and citrulline are cut out and counted in a liquid scintillation spectrophotometer. The method has the following advantages: (1) the radioactive substrate ornithine is more stable in solution than carbamoyl phosphate, (2) 14C-labeled ornithine is available in higher specific activity than carbamoyl phosphate, (3) all radioactivity may be accounted for by using the TLC system, (4) the developed thin-layer chromatogram is stable indefinitely, (5) in contrast to colorimetric assays, other compounds in the raction mixture do not interfere with the citrulline determination, and (6) most importantly, the rate of the enzyme reaction at various time intervals can be determined by taking aliquots from the same incubation tube.     

Detection of cardiac-specific creatine kinase isoenzyme in sera with normal or slightly increased total creatine kinase activity.

We describe a spectrophotometric kinetic assay for detecting creatine kinase MB isoenzyme activity in the 1 to 10 U/liter range. The MB isoenzyme was isolated [Clin. Chem. 20, 36 (1974)] and assayed (Rosalki method) with an Abbott ABA-100. Good reproducibility was demonstrated for MB isoenzyme activities near 1 U/liter (CV = 2.6%). Sera with normal or slightly increased total creatine kinase activity were evaluated. Sera of 14 patients with acute myocardial infarction contained, per liter, 84 to 236 U of total creatine kinase activity and 4.6 to 28.0 U of isoenzyme MB activity; corresponding ranges for sera from healthy lab technicians and patients with noncardiac disease were 36 to 277 and 0 to 2.6 U. MB isoenzyme activity for infarction patients rose and fell sharply within three days after the infarction. Atypical time-course patterns, MB isoenzyme activity remaining abnormally great for five days, were observed in serum from patients with prolonged atrial fibrillation and congestive heart failure or cardiomyopathy; the BB isoenzyme (1 to 5 U/liter) was also detected in sera of such patients but was absent in sera from infarcation patients. Quantification of column-isolated MB by the assay described is rapid, easy, specific, and extremely sensitive for measuring MB in the 1 to 10 U/liter range.     

Sensitive, rapid assay of subforms of creatine kinase MB in plasma

The subforms of creatine kinase (CK; EC 2.7.3.2) in plasma have received recent attention as potential markers for the early diagnosis of acute myocardial infarction. Because changes in CK-MM subforms are not specific for myocardial injury, we developed an assay, based on high-voltage electrophoresis, that is sufficiently sensitive to detect the CK-MB subforms at concentrations substantially below the upper limit of normal (14 U/L). The assay can detect 1.25 U of either MB subform per liter with a precision of 0.20 U/L and gives responses that vary linearly with activity concentration from 0.0 through 30.0 U/L, with an identical signal response for both subforms. When both subforms are present in a serum sample, the assay accurately measures both the relative percentage and the absolute quantity of each: assay activity/known activity was 1.03 for each subform at a total MB subform activity of 5.0 U/L (r = 0.98). Assay time is 25 min, and there is no loss of CK during electrophoresis. Thus, this system can be used to rapidly, sensitively, and precisely quantify the two CK-MB subforms at activities well within the normal reference interval.     

Lysinuric protein intolerance. Basolateral transport defect in renal tubuli.

In patients with an autosomal recessive diamino acid transport disorder, lysinuric protein intolerance (LPI), we measured plasma and urinary amino acids basally, and during intravenous infusion of citrulline at two rates. Compared with controls, the patients' plasma citrulline concentrations rose similarly, but urinary citrulline excretion increased excessively. Their plasma arginine and ornithine levels rose subnormally, but massive argininuria and moderate ornithinuria appeared. The excretion rates of the third diamino acid lysine and other amino acids remained practically unaltered, thus excluding mutual competition as the cause for the increases. The results suggest that (a) in the normal kidney reabsorption involves partial conversion of citrulline to arginine and ornithine (metabolic run-out), (b) in LPI, the diamino acid transport defect is located at the basolateral cell membrane of the renal tubules; this inhibits the efflux of arginine and ornithine, increasing their cellular concentration, which in turn inhibits the metabolic disposal of citrulline, and causes leakage of arginine, ornithine, and citrulline into the tubular lumen.     

Direct spectrophotometric determination of alpha-amylase activity in salive, with p-nitrophenyl alpha-maltoside as substrate.

We describe a simple, direct kinetic method for determination of salivary alpha-amylase (1,4, alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1). The assay makes use of a well-defined substrate, p-nitrophenyl alpha-maltoside, which is hydrolyzed by alpha-amylase to a chromogenic product, p-nitrophenol. Activity is determined by directly monitoring the increase in absorbance of the reaction mixture. Amylase activity can be defined in international (IUB) units of micromoles of product/min per liter of saliva. For 22 healthy subjects, the mean +/- SD of amylase activity in mixed saliva was 2.77 +/- 1.12 U/liter. Activity and instrumental response were linearly related over the entire range tested (0.224 to 11.90 U/liter). The within-run precision (CV) over this range was better than 3% for all but the lowest activities. Values obtained with this assay correlate well with those obtained with a modified Nelson-Somogyi saccharogenic method (r = 0.979). The precision and simplicity of this assay suggest that it is the method choice for determining amylase activity in human saliva.     

Investigation of urea cycle enzyme disorders by 1H-NMR spectroscopy.

High resolution proton nuclear magnetic resonance spectroscopy (1H-NMR) has been used to study patients with inborn errors of the urea cycle to evaluate further the diagnostic potential of this technique. The 1H-NMR metabolic profile from the urine of patients with citrullinaemia and argininosuccinic aciduria consistently demonstrated the presence of the diagnostic metabolites citrulline, N-acetylcitrulline and argininosuccinate, respectively. The profile from the urine of patients with ornithine carbamoyl transferase deficiency, is potentially diagnostic, but orotate was only detected in samples from three out of four patients. The characteristic fingerprint that each of the metabolites produces is unlike that of any other we have seen, including analogues of the metabolites which are structurally very similar such as arginine, ornithine and aspartate. The level of excretion of the metabolites from the patients with citrullinaemia and argininosuccinic aciduria has been well within the range of NMR detection.     

Direct micromethod for colorimetry of serum ornithine carbamoyltransferase activity, with use of a linear standard curve.

Determination of serum ornithine carbamoyltransferase (EC 2.1.3.3) activity can be a valuable diagnostic tool in the detection of liver diseases involving cytolytic processes. I describe a micromethod for measuring this activity in serum, in which the reaction product, citrulline, is measured colorimetrically in the incubation mixture without prior deproteinization. To eliminate the interference of serum protein precipitation, the concentration of sulfuric acid in the color reagent has been decreased, without substantial loss of sensitivity. Optimizing the conditions of citrulline determination, in which antipyrine and 2,3-butanedione monoxime are used, has resulted in a linear standard curve. The color formed by citrulline is found to be stable in room lighting and sensitive only to direct sunlight. The precision of the method is inversely correlated to serum enzyme activity, the CV varying between 4.6 and 21.1%.     

Heterogeneity of patients with late onset ornithine transcarbamylase deficiency.

Fourteen patients, 10 males and 4 females, with "late onset" ornithine transcarbamylase (OTC) deficiency were diagnosed by enzyme assays performed on their liver tissues. Age of first clinical presentation ranged widely from 10 weeks to 23 y (mean = 6.1 y). Peak plasma ammonia levels varied widely from a low of 105 mumol/L to as high as 800 mumol/L. All patients had elevated plasma levels of glutamine whereas plasma levels of citrulline were normal in 6 patients. Plasma ornithine levels were not elevated in any patient. Orotic aciduria of variable degree was detected in 13 patients. Residual hepatic OTC activity was detectable in 13 out of the 14 patients ranging from 0.7 to 28.3 mumol/g/min (normal = 80.6 +/- 19.1, mean +/- SD, n = 52). Ten patients were alive at the time of this report and 5 of them had psychomotor delay.     

Rapid quantitative, specific measurement of pancreatic amylase in serum with use of a monoclonal antibody

In this rapid quantitative assay for pancreatic alpha-amylase (EC 3.2.1.1) in serum, we precipitate salivary amylases by 10-min incubation with monoclonal anti-salivary amylase antibody immobilized on particles of polyvinylidene fluoride. We then centrifuge the serum mixture and measure the pancreatic amylase activity remaining in the supernate by a kinetic method. The assay requires 50 microL of serum and the standard curve is linear to at least 1300 U of pancreatic amylase per liter of serum. CVs were 1.3% within-run, 6-8% day-to-day. Apparent analytical recovery of pancreatic amylase activity added to serum was 101% +/- 2%. Addition of purified salivary amylase, 356 U/L, to sera gave a value for apparent pancreatic amylase of less than 4 U/L, or 1% of the added salivary amylase activity. This assay correlated well with an electrophoretic method (slope, 0.97-0.99; intercept, 0.5 to -4 U/L; correlation coefficient, 0.946-0.990; and standard error of the estimate 3-5 U/L). Estimated normal reference intervals with maltotetraose as substrate were: total amylase, 39-118 U/L; pancreatic amylase, 11-50 U/L; and salivary amylase, 18-79 U/L.     

Reaction-rate assay of serum sorbitol dehydrogenase activity of 37 degrees C.

The sorbitol dehydrogenase (EC 1.1.1.14) activity of human serum and liver was investigated at 37 degrees C. Various buffers were examined but tris(hydroxymethyl)aminomethane HCI in a final concentration of 90 mmol/liter (pH 6.6, at 37 degrees C) was the one with which activity was the greatest. The enzyme is inhibited by its substrate, D-fructose, and activity was greatest with a substrate concentration (in the final assay mixture) of 500 mmol/liter and an NADH concentration of 247 mumol/liter. The specimen volume was 100 mul. The enzyme from liver and serum was shown to have similar Km values and activation energies, and we conclude that the liver enzyme was unchanged on passing into the extrahepatic space. Normal values for this assay are 0--2.6 U/liter (2.5 and 97.5 percentiles).     

Early alterations of plasma free amino acids in chronic renal failure

In order to assess the influence of renal failure and nutritional status on the fasting concentrations of free plasma amino acids, we studied 81 ambulatory adult patients with varying degrees of chronic renal failure. Each of the patients was in good general and nutritional condition. Compared to 33 healthy controls, patients with mild renal failure (Ccr greater than 25 ml/mn) exhibited significantly (p less than 0.01, Student's t test) raised concentrations of cystine, citrulline, ornithine, taurine and 3-methyl-histidine and low level of serine. Concentrations of cystine, citrulline, and 3-methyl-histidine in plasma but not of taurine or ornithine rose in parallel with the progression of renal failure. A significant, but moderate decrease in valine, leucine and isoleucine concentrations was observed in patients with the most marked degree of renal failure (Ccr less than 10 ml/mn). We conclude that changes in the plasma concentration of several non essential amino acids are already present in the early stage of renal failure in patients with no sign of protein malnutrition: these may result from altered metabolic pathways of amino acids related to uremia and/or nephron loss per se whereas the moderate decrease in branched-chain amino acids that is observed only in the advanced stage of renal failure may be, at least in part, nutritional in origin.     

乙型肝炎病毒相关慢性肝炎、肝硬化和慢加急性肝衰竭患者血浆氨基酸谱变化及特点

目的分析乙型肝炎病毒（HBV）相关慢性肝炎、肝硬化、慢加急性肝衰竭患者血浆氨基酸谱的变化及特点,为肝病严重程度判断和临床针对性干预提供依据。方法选择35例HBV相关慢性肝病包括慢性乙型肝炎、乙型肝炎肝硬化、慢加急性肝衰竭患者和10例健康对照者,应用质谱方法检测其血浆氨基酸、肝功能和血氨,分析以上不同类型肝病氨基酸谱变化及其特点,同时观察尿素循环中重要氨基酸代谢产物瓜氨酸和鸟氨酸水平与血氨的变化。结果与正常对照比较,慢性乙型肝炎患者尚无明显氨基酸代谢失衡;肝硬化和慢加急性肝衰竭患者血浆芳香族氨基酸水平升高,支链氨基酸/芳香族氨基酸比值明显降低。慢性乙型肝炎、肝硬化和肝衰竭患者血浆鸟氨酸、瓜氨酸、血氨均较正常对照组升高。结论慢性乙型肝炎患者血浆氨基酸谱尚无明显变化,一旦发展到肝硬化和慢加急性肝衰竭则变化明显,尤其支链氨基酸/芳香氨基酸比值,鸟氨酸、瓜氨酸与血氨变化显著。基于血氨升高和氨基酸失衡在肝性脑病发病中的重要作用,本研究结果再次提示,血浆氨基酸谱、鸟氨酸、瓜氨酸与血氨检测可以用于肝病严重程度判断、肝性脑病的预测,并指导临床治疗。     

A therapeutic, highly purified factor XI concentrate from human plasma

A highly purified factor XI (FXI) concentrate was prepared from human plasma by a process comprising a filter adsorption step and chromatography on a cation exchange resin. The freeze-dried FXI, which solubilized quickly, had high specific activity (130-150 U/mg protein), high potency (approx. 100 U/mL), and excellent stability for at least 24 hours at room temperature in the liquid state. The overall recovery was about 220 U of FXI per liter of plasma. Minor protein contaminants (C1-inhibitor, fibronectin, IgG, and alpha-2-macroglobulin) were found to be between 0.13 and 0.46 mg per 1000 U of FXI. Fibrinogen and relevant coagulation factors (factors II, V, VII, IX, X, XII, XIII, and VIII/von Willebrand factor) were undetectable, as evidenced by immunologic and immunoelectrophoretic data. Components of the kinin system were present in trace amounts or were undetectable. No evidence of activated factors such as factors Xa and IXa was found. Proteolytic activity, as assessed by S-2288 chromogenic substrate, was negligible and thrombin was undetectable. A solvent-detergent treatment was included prior to chromatographic purification to enhance viral safety against lipid-enveloped viruses. In vitro and in vivo animal studies demonstrated the absence of thrombogenic, hypotensive, or toxic effects. No thrombogenic activity was found in the Wessler model in rabbits at doses of 900 to 1100 U of FXI per kg of body weight. This FXI preparation could be beneficial in substitution therapy of congenital or acquired FXI deficiency, especially as a way to avoid the use of fresh-frozen plasma.     

Kinetic determination of serum sorbitol dehydrogenase activity with a centrifugal analyzer

We describe a mechanized method for centrifugal analyzer determination of sorbitol dehydrogenase in serum, based on conversion of D-fructose to sorbitol with simultaneous oxidation of NADH, in triethanolamine buffer at pH 7.4 and 30 degrees C. The standard curve for this assay is linear to 200 U of activity per liter of serum. The mean within-run precision (CV) of the assay is 0.8%. Results correlate well with those by a spectrophotometric method. In sera from 20 apparently healthy adult humans, sorbitol dehydrogenase activity averaged 1.7 (SD +/- 0.8; range, 1-3) U/L. The mean activity (U/L) for a group of 30 rats was 4.4 (SD, +/- 0.2; range, 3-6); for 20 dogs, 5.8 (SD, +/- 0.7; range 3-9); and for 30 mice, 26.8 (SD +/- 2.1; range, 22-34). To determine the utility of measuring this enzyme in the serum of rats for assessment of hepatotoxicity in drug-safety studies, we compared sorbitol dehydrogenase activity with that of alkaline phosphatase, aspartate aminotransferase, and alanine aminotranferase in the sera of rats treated with thioacetamide or in which the common bile duct has been ligated.     

Significance of arginase and ornithine in malignant tumors of the human skin

During neoplastic development, several aspects of the regulation of polyamine synthesis undergo profound changes. In extrahepatic mammalian tissues in which the urea cycle is not functioning, arginase is believed to supply the cell with ornithine, a non-protein amino acid that is a precursor for biosynthesis of polyamines. Because the activity of ornithine decarboxylase and polyamine levels have been shown to be elevated during carcinogenesis, we decided to investigate the role of arginase in the development of malignant tumors of the human skin and to examine whether arginase activity and ornithine level can be used as biologic markers for distinguishing patients with squamous cell cancer from patients with basal cell cancer. For this purpose, we studied tissue arginase activity and ornithine level in tumor and adjacent normal tissues in 16 patients (55 +/- 10 years of age) with malignant skin tumors (8 of which were squamous cell cancers and 8 of which were basal cell cancers). The mean arginase activity and ornithine levels in tumor tissues (total) were 17.75 +/- 8.54 U/mg protein and 40.89 +/- 14.88 nmol/mg protein, respectively, versus 3.69 +/- 1.71 U/mg protein and 12.98 +/- 6.21 nmol/mg protein, respectively, for normal tissues. The mean specific arginase activity levels in squamous cell and basal cell cancers of the human skin were 18.49 +/- 10.47 U/mg protein and 16.63 +/- 6.00 U/mg protein, respectively. The mean ornithine levels in squamous cell and basal cell cancers of the human skin were 42.45 +/- 19.10 nmol/mg protein and 39.33 +/- 10.19 nmol/mg protein, respectively. Our results indicated that (1) arginase activity and ornithine levels are elevated in squamous cell and basal cell cancers of the human skin; (2) the increased activity of arginase and hence the elevated levels of ornithine may be important in the development of malignant tumors of the human skin; and (3) although arginase activity and ornithine level may be useful for distinguishing patients with malignant skin tumors from healthy subjects, they cannot be used as biologic markers for distinguishing patients with squamous cell cancer from patients with basal cell cancer.     

Measurement of Plasma Renin Concentration (PRC) Using Exogenous Substrate and Radioimmunoassay

Abstract. An assay to determine renin concentration (RC) has been developed which allows quantitation on a scale of Goldblatt units (G.U.) and provides a way of comparing results from various laboratories. Renin activity is protected by Cleland's reagent. The enzymatic reaction is standardized for pH (8.0), temperature (30^o C), time (30 min.) and by using hog plasma renin substrate (optimal pH 8.0). The latter avoids the interference of pseudorenin (optimal pH 5.0) and reduces the effects of endogenous renin substrate upon the enzyme reaction. The standard curves were corrected for the blank. Angiotensin I generated was measured by a double antibody radioimmunoassay method. Recoveries of added enzyme provided a control for the assay and were 96±12.5% (SD) for 47 experiments. The coefficient of variation was 16.8% and the sensitivity of the assay (=2 SD) was 0.2×10^-4 G.U. per ml plasma. The effect of various materials on the hydrolysis of hog substrate by renin was studied. Plasma renin concentration (PRC) from healthy individuals on an uncontrolled diet and in the upright position was 0.88 ±0.09 times 10^-4 G.U. per ml. Patients suffering from primary aldosteronism had suppressed values of PRC which reached maximal levels of 0.2 ×10^-4 G.U. per ml in the upright position on a sodium restricted diet (10 mEq per day).     

Enzymatic determination of l-ornithine in serum

Serum l-ornithine was determined enzymatically using ornithine carbamoyltransferase (OCT) from Streptococcus faecalis and measuring the citrulline formed with a diacetyl-thiosemicarbazide reaction after precipitation of proteins with trichloracetic acid. The normal fasting values, 26–92 μmol/1, correlated well with those obtained by Chromatographic methods.     

Radioenzymatic assay for direct measurement of plasma pyridoxal 5'-phosphate

A radioenzymatic assay for measurement of pyridoxal 5'-phosphate (PLP) is described, based on the incubation of L-[3H]tyrosine (10(6) cpm, spec. acty. 1.88 Ci/mol) in the presence of the apoenzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis and PLP in phosphate buffer (0.1 mol/L, pH 5.5) at 37 degrees C for 60 min. The decarboxylated metabolite formed, [3H]tyramine, was selectively extracted into ethyl acetate, and the tritium radioactivity in the sample was determined by liquid scintillation counting. As little as 0.5 nmol of PLP can be detected per liter. The assay is specific, no cross reactivity having been noted for several compounds closely related to PLP. With this we could directly measure the concentrations of PLP in plasma without prior deproteinization and ether washing of the samples. Using the assay to determine plasma concentrations of PLP in healthy adult populations, we found results that were comparable with previously reported data.     

Sensitive assay of creatine kinase isoenzymes in human serum using M subunit inhibiting antibody and firefly luciferase

A sensitive method for the assay of B subunit and total creatine kinase activity is described. The ATP formation in the creatine kinase reaction is continuously monitored by measuring the bioluminescence obtained with a purified firefly luciferase reagent. The B subunit activity is determined using an M subunit inhibiting antibody resulting in greater than 99.5% and approximately 50% inhibition of MM and MB isoenzymes, respectively. The bioluminescent method correlated well with a similar spectrophotometric method in the assay of B subunit as well as total creatine kinase activity (r greater than or equal to 0.98). However, the bioluminescent assay is considerably more sensitive, allowing the assay of B subunit activity in serum from healthy individuals. This is due to the inherent sensitivity of the bioluminescent assay of ATP, a reduced analytical interference from adenylate kinase and a reduced reagent blank. The within-series precision at 1 U/liter and greater than 52 U/liter corresponded to a C.V. of 14% and 3%, respectively. The method is as rapid and suitable for routine work as spectrophotometric methods. From a clinical point of view the new method is of particular interest in the early diagnosis of small acute myocardial infarctions.