Serum ammonia 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. 

The determination of ammonia in blood is carried out enzymatically, which is considered to be specific, precise and simple. (48) Serious mistakes can easily occur during the preanalytical phase of ammonia determination, making it imperative to comply with the standardized method of taking a blood sample, (s. p. 91) EDTA blood should be taken with the addition of sodium borate and L-serine. Furthermore, elevated serum y-GT activity and increased thrombocytes cause the ammonia level to rise, as does cigarette smoking prior to blood collection. Even minor haemolysis (e. g. in the event of prolonged transport) will spoil the blood for ammonia determination, since the ammonia concentration of erythrocytes is three times that found in plasma. Besides these interfering factors, ammonia concentration is influenced by (7.) the metabolic performance of the urea cycle, (2.) the extrahepatic formation and elimination of ammonia, and (5.) the acid-base status. 

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. 

The concept of a biocatalytic membrane electrode has been extended to the use of a tissue slice as the catalytic layer. An example of this approach is an electrode for AMP which consists of a slice of rabbit muscle adjacent to an ammonia gas electrode. NHj is produced by enzymatic action of rabbit muscle constituents on AMP The electrode exhibits a linear range of 1.4 x 10 to 1.0 x 10 M with a response time varying from 2.5 to 8.5 min, depending on the concentration. Electrode lifetime is about 28 days when stored between use in buffer with sodium azide to prevent bacterial growth. Excellent selectivity enables AMP to be determined in serum. 

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. 

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... 

A method has been published for the determination of a variety of metals in diluted blood and serum using inductively coupled plasma atomization with mass spectrometric detection.3 Blood was diluted tenfold and serum fivefold with a solution containing ammonia, Triton X-100 surfactant, and EDTA. Detection limits adequate for measurement in blood or serum were found for cadmium, cobalt, copper, lead, rubidium, and zinc. 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

Ammonia/Urea Serum, urine Biochemical Conventional Sequential determination. 150... 

Although delays of a specimen in transit from a patient in a hospital to the laboratory are usually short, the time elapsing from the separation of serum and cells until analysis may be considerable. The specimen must be properly treated both during its transport to the laboratory and from the time the serum has been separated until it is analyzed. For some tests, specimens must be kept at 4 C from the time the blood is drawn mitil the specimens are analyzed, or until the serum or plasma is separated from the cells. Examples are specimens for ammonia and blood gas determinations, such as PCO2, PO2, and blood pH (see Chapter 27). Transfer of these specimens to the laboratory must be done by placing the specimen container in ice water. Specimens for acid phosphatase, lactate and pyruvate, and certain hormone tests (e.g., gastrin and renin activity) should be treated the same way. A notable decrease in pyruvate and increase in lactate concentration occurs within a few minutes at ambient temperature (see Chapter 25). 

Another ammonia sensor speciHcally designed for use in bioliquids is based on the evanescent wave technique and can be applied to the vapor-phase determination of ammonia above blood and serum [136]. It utilizes the ninhydrin reaction occurring in the polymer coating of the fiber, and the resulting color change is monitored by total internal reflection. The probe is applicable to clinical determinations normally carried out in the vapor phase, but works irreversibly. A linear relationship exists between absorbance and ammonia concentration in the clinically useful range of 0-4.0 pg mL. Comparison with the reference method showed a correlation coefficient of 0.92. 

B. Other useful laboratory studies include electrolytes, glucose, BUN, creatinine, calcium, ammonia, liver transaminases, bilirubin, prothrombin time (PT), amylase, serum osmolality and osmolar gap (see p 32 serum levels > 1500 mg/L may increase the osmolar gap by 10 mOsm/L or more), arterial blood gases or oximetry, and EGG monitoring. Valproic acid may cause a falsepositive urine ketone determination. 

Creatinine The microbial enzyme creatinine deimi-nase (EC 3.5.4.21) works well in the enzyme thermistor giving a linear range from 0.01 to at least 10 mmol 1 This could be a very powerful assay for clinical use since it requires only one enzyme and is independent of endogenous ammonia. Unfortunately, the determination of creatinine in human serum requires a higher order of magnitude increase in sensitivity than is available with the present instrumentation. 

Because of the high selectivity and sensitivity of the postcolumn fluorescence detection of histidine with OPA, the present HPLC method is applicable to a specific and rapid assay of histidine in human serum, blood, and urine after simple pretreatment. A recent paper demonstrated that the postcolumn detection with OPA was applicable to the simultaneous assays of histidine and its major metabolites cis- and frawi-urocanic acids) in human stratum cor-neum. -" The postcolumn detection system was also applicable to the flow injection analysis (FIA) method for the assay of histidine in serum and urine. The FIA method enabled us to determine histidine in blood after pretreatment of the sample with A-ethylmaleimide (masking reagent of glutathione).These methods are useful in the diagnosis of histidinanemia, one of hereditary metabolic disorders characterized by a virtual deficiency of histidine ammonia-lyase. 

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