Serum urea determination

The above discussion does not mean that the use of urease and subsequently the use of an ammonia electrode is not practicable for a urea determination. Unfortunately, the commercial company that produced the urea analyzer, chose a conductivity procedure, which happens to be unsuitable for the laboratory of Neonatology. Had they chosen the ammonia electrode, which happens to be a relatively good electrode, and is especially specific, since only ammonia and not potassium can pass an air space, then the instrument could have been made highly specific for urea. In this case an ammonia determination would be done initially and then subtracted by the computer, from the amount which has been generated subsequently. In any case, with present technology, sensitivity is not adequate to use less than approximately 15fil of serum. 

Welch MJ, Cohen A, Hertz HS, Ruegg FC, Schaefer R, Sniegoski LX and White VE (1984) Determination of serum urea by isotope dilution/mass spectrometry as a candidate definitive method. Anal Chem 56 713-719. 

Number Serum Urea Concentration (mM) Urea Concentration Determined (mM) Relative Standard Deviation (%)... 

Rovida E, Mosca A, Dossi G. Serum and whole blood urea determination by the use of a microprocessor controlled differential pH analyzer. Eur J Chn Chem Chn Biochem 1981 19 820. 

Diagnosis of renal problems, xanthinuria, and toxemia of pregnancy via determination of the ratio of hypoxanthine to xanthine in plasma is facilitated by the use of biosensors. Xanthine oxidase immobilized on aminopropyl-CPG (controlled pore glass) activated with glutaraldehyde oxidizes hypoxanthine first to xanthine and then to uric acid. Use of an IMER with biosensors for hypoxanthine, xanthine, and uric acid provides the necessary data. Pre- or postcolumn enzymatic reactions catalyzed by creatinine deiminase, urease, alkaline phosphatase, ATPase, inorganic pyrophosphatase, or arylsufatase facilitate analysis of uremic toxins (simultaneous detection of electrolytes, serum urea, uric acid, creatinine, and methylguanidine). 

Amino acids constitute the second largest source of nonprotein nitrogen in serum, urea being major source. The determination of total serum amino acids can provide valuable clinical information. Single amino acids are measured to gain access to particular enzyme activities of transaminases and peptidases for instance. Amino acids are also important in the food industry and in biotechnology. Their concentration in food can be used as a measure of the nutritive value of the food. 

Hamann (1987) employed a potentiometric urea electrode in an enzyme difference analyzer for urea determination in serum. The difference between the potential changes of a urease-covered and a bare pH glass electrode is evaluated 30 s after sample injection. This fixed-time regime provides a measuring frequency of 20-25/h the linear range for 1 120 diluted samples is 1-20 mmol/l. These results are better than those of common potentiometric enzyme sensors. 

Figure 8 NIR-predicted serum urea and serum protein levels compared to reference anal5hical results (see also NIR A in Table 4). The line of identity is included. (Adapted by permission of Elsevier Science from J.W. HaU, A. PoUard, Near-infrared Spectroscopic Determination of Serum Total Proteins, Albumin, Globulins, and Urea , Clinical Biochemistry, 483-490, Vol. 26, 1993 by the Canadian Society of Clinical Chemists.)...

Determination of Serum Urea by Mass Fra gmen t o graphy... 

Marsh, W. H. Fringerhut, B. and Miller, H. (1965). Determination of serum urea by diaoxal method. In Practical clinical biochemistry, 5 (edn) by Varley London. 

Martinez-Eabregas, E. Alegret, S. A Practical Approach to Ghemical Sensors through Potentiometric Transducers Determination of Urea in Serum by Means of a Biosensor, /. Chem. Educ. 1994, 71, A67-A70. 

Directions are provided for constructing and characterizing an ammonium ion-selective electrode. The electrode is then modified to respond to urea by adding a few milligrams of urease and covering with a section of dialysis membrane. Directions for determining urea in serum also are provided. 

In the particular example shown, zinc sulfate and barium hydroxide are being dispensed into the test tube so as to precipitate the proteins. The filtrate obtained is the filtrate from 10 microliters of serum. This can be used for several purposes and in the application being referred to, an amount equivalent to 3 microliters is being used for sugar determination, by the hexokinase procedure and an amount equivalent to 3 microliters is being used for urea estimation with diacetylmonoxime (15). 

Recently, the old alkaline phenol method has been revived, and is being widely used in clinical laboratories, without protein preclpltatlon(27). In this procedure, the serum is added to an alkaline phenol reagent, and the ammonia generated from urea is determined either after the action of urease or after strong alkaline treatment of the serum. The objection to this procedure is first, that all urease is rich in ammonia, and second, the color produced with alkaline phenol is not specific for ammonia. It will react with other compounds, especially for those that liberate ammonia. By this procedure one obtains a useful number from the point of view of determining whether the patient has nitrogen retention, but a value which is somewhere between a urea and an N.P.N. determination. 

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

Nitrogen compounds commonly determined are creatinine, urea, and uric acid. Creatinine is an end product of the energy process occurring within the muscles, and is thus related to muscle mass. Creatinine in urine is commonly used as an indicator and correction factor of dilution in urine. Creatinine in serum is an indicator of the filtration capacity of the kidney. Urea is the end product of the nitrogen luea cycle, starting with carbon dioxide and ammonia, and is the bulk compoimd of urine. The production of uric acid is associated with the disease gout. In some cases, it appears that the excess of uric acid is a consequence of impaired renal excretion of this substance. 

Patients should have blood urea nitrogen, serum creatinine, aspartate transaminase or alanine transaminase, and a complete blood count determined at baseline and periodically, depending on the presence of other factors that may increase the likelihood of toxicity (advanced age, alcohol abuse, and possibly pregnancy). Hepatotoxicity should be suspected in patients whose transaminases exceed five times the upper limit of normal or whose total bilirubin exceeds 3 mg/dL. At this point, the offending agent(s) should be discontinued, and alternatives selected. 

Examples of the use of FIA with ISE detection involve the determination of nitrate and total nitrogen in environmental samples [48, 49, 125, 166], potassium, sodium [125], calcium [51] and urea [124] in serum or major nutrients in fertilizers [73]. An interesting combination of an ISFET sensor with the FIA principle [52] is shown in fig. 5.17. This is a simultaneous determination of potassium, calcium and pH in serum during dialysis on an artificial kidney. 

Among potentiometric enzyme sensors, the urea enzyme electrode is the oldest (and the most important). The original version consisted of an enzyme layer immobilized in a polyacrylamide hydrophilic gel and fixed in a nylon netting attached to a Beckman 39137 glass electrode, sensitive to the alkali metal and NHj ions [19, 2A Because of the poor selectivity of this glass electrode, later versions contained a nonactin electrode [20,22] (cf. p. 187) and especially an ammonia gas probe [25] (cf. p. 72). This type of urea electrode is suitable for the determination of urea in blood and serum, at concentrations from 5 to 0.05 mM. Figure 8.2 shows the dependence of the electrode response... 

Lalezari and Lalezari synthesized urea derivatives of bezafibrate, and, with Perutz, determined the binding site of the most potent derivatives to be the same as that we had discovered for bezafibrate [35], Although all of these compounds were extremely potent, they were not suitable clinical candidates on account of being hampered again by serum protein binding [36, 37]. 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

---