Evaluating Novel Markers for Specimen Validity Testing.

CONTEXT.­: Synthetic urine products are commercially marketed for the purpose of specimen substitution for urine drug screens. These products are widely popular because they yield negative drug screen results, meet criteria for specimen validity testing, and are easily accessible and affordable. Current specimen validity criteria are ineffective for detecting these synthetic products, and new markers of specimen validity are required. OBJECTIVE.­: To develop and evaluate a multicomponent liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for urine specimen validity testing. DESIGN.­: A quantitative LC-MS/MS assay was developed for caffeine, cotinine, theobromine, and urobilin in urine. The assay was applied to known synthetic urine products (n = 10) as well as human specimens received for pre-employment testing (n = 500), for-cause workplace testing (n = 100), and medical pain management monitoring (n = 200). Specimens devoid of all 4 validity markers were subjected to follow-up testing that involved microscopic urinalysis and comprehensive gas chromatography mass spectrometry for drugs, pharmaceuticals, hormones, and lipids. RESULTS.­: Of the experimental groups, 10 of 10 synthetic urine products (100%), 12 of 500 pre-employment specimens (2.4%), and 4 of 200 pain management specimens (2.0%) failed the experimental LC-MS/MS assay. Follow-up testing indicated that each of the failed specimens was nonphysiologic in nature. CONCLUSIONS.­: Simultaneous application of the 4 experimental validity markers appeared to be a robust method for detecting nonphysiologic specimens. New markers of specimen validity must be developed in order to identify commercially available synthetic urine products.

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

[Metabolism of bile pigments in the intestine]. / Le devenir des pigments biliaires dans l'intestin.

This review discusses the knowledge on the transformation of bilirubin into urobilinoids before its elimination from the organism. The following aspects are presented: mechanism of bilirubin reduction by anaerobic intestinal bacteria, structure of the resulting urobilinoids, and enterohepatic circulation of the latter. Investigations on the reduction of bilirubin by intestinal bacteria in vitro are described, and the diagnostic value of urobilinoids determination is discussed.

Interrelationships between clinical medicine and clinical chemistry, illustrated by the example of the German-speaking countries inthe late 19th century.

The development of Clinical Chemistry in the German-speaking countries in the second half of the 19th century is characterized by a close relationship between Clinical Medicine and Clinical Chemistry. Clinical Chemistry lost its relatively independent basis and was performed by clinicians themselves. Using examples, it is shown that the new concept of "pathological physiology" led to the adoption of clinical chemistry as an area of clinical research. As a result of this development laboratories were established in hospitals, and clinical-chemical examinations were used in clinical practice more than before. Even medicine itself, e.g., nosography was influenced by this development.

[Bilirubin in the blood serum of clinically healthy cows and those ill with ketosis and hepatopathies]. / Bilirunbinut v kruvniia serum na kravi, klinichno zdravi, bolni ot ketoza i s khepatopatii.

Studied was the bilirubin equilibrium in 62 normal cows, 52 cows with clinical ketosis, 31 cows with diffuse liver injury, and 14 cows that went off food for five days. A moderate rise of the total bilirubin of mixed type was found in ketosis-affected cows. Statistically significant proved the changes in the free fraction, however, there were no persuasive data that they were due to superproduction of bilirubin. Changes in the total bilirubin similar to those in cows with ketosis were established also in cows subjected to starvation, substantiated by the adequate rise of the free and the bound fraction. The hyperbilirubinemia in cows with diffuse liver injuries was found to be of a mixed type, with a very clear transposition in the bilirubin fractions. The direct: indirect ratio of bilirubin in this case was higher than unit, and the percent participation of the indirect fraction was brought down to 42.5. Apparently, the symptoms of liver affections were beyond the frames of what was found in the cows with ketosis. With the use of tests for the demonstration of urobilin and urobilinogen in the urine no dependable idea could be found of the status of the pigment fraction in the liver of ketosis-affected cows or the fact that this fraction was involved to a considerable extent. Discussed is the possibility of linking the state of hyperbilirubinemia in cows with ketosis with some energy deficiency with regard to the plasma glucosis and the liver glycogen.

[Determination of urobilinoide concentration in the urine. New rapid method for clinical determination of urinary-urobilinoids using the Ehrlich-reaction]. / Bestimmung der Urobilinkörper-Konzentration im Harn Eine neue Schnellmethode zur klinischen Bestimmung der Harn-Urobilinoide mit der Ehrlich-Reaktion

A rapid method for quantitative determination of concentration of urobilinoids of fresh urine was developed. The method is based upon extraction of urinary urobilinoids using a mixture of acetic acid and ether and followed by the extraction of the ethereal phase with Ehrlich reagent and a solution of sodium acetate. Readings of absorbance from the absorption band of reaction products from urobilinoids and Ehrlich reagent are done. Performance of the rapid method: 2 ml urine + 10 ml ether + 2 ml acetic acid were thoroughly mixed for about 15 seconds. The ethereal extract was shaken after addition of 12 ml Ehrlich-reagent for 1 minute and 15 ml half-saturated sodium acetate solution were added. 0-3 minutes afterwards reading of absorption at 560 nm respectively filter Hg 578 nm. Photometer:mg/dl=29 X A578 nm, 1 cm -0,4. Spectrophotometer:mg/dl urobilinoids=13,7 X A560 nm X 1/d -0,22 (d=optical pathway). Photometrical determination of concentration of urobilinoid can also be done using table 1. Accuracy, reproducibility and specificity are fully satisfying for clinical use of the method.

[Several functional changes in the livers of patients with diabetes mellitus]. / Vyrkhy naikoi funktsionalni chernodrobni promeni pri bolni sus zekharen diabet

The authors have studied the functional liver state in 107 patients with diabetes mellitus. Various stages of functional liver disturbances were found in a considerable part of them. With a few exceptions, the latter have the highest incidence and severity among the patients with decompensated, with mixed type diabetes and with remote vascular degenerative manifestations. Radioisotope investigations with 131J-Bengal pink, reveals the earliest developed changes in some of the liver functions, as compared with rest of the tests. The functional liver state is recommended to be determined in all diabetics with a view to an early detection of the developed functional disturbances and their timely treatment.

Biochemical monitoring of the surgical patient.

The need for prognostic and biochemical monitors of cellular function becomes increasingly evident with the further elucidation of the metabolic pattern secondary to disease. Thus far studies have shown that lactic acid may be one of these biochemical parameters. Investigations are now in the process of defining other biochemical indicators of cellular dysfunction.

Urinary estrogen assays and maternal hyperbilirubinemia.

Urinary estrogen determinations have simplified the care of pregnant patients with certain metabolic disorders known to adversely affect the fetus. This report demonstrates that erroneously low values for urinary estrogens may be obtained if colorimetric assay methods are used for patients with congenital hemolytic anemias during an acute hemolytic crisis. This appears to be due to the interference of the large amounts of urobilin present in the urine of these patients.