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A major concern with the identification of renal toxicity using the traditional biomarkers, urea and creatinine, is that toxicity signal definitions are not sensitive to medically important changes in these biomarkers. Traditional renal signal definitions for urea and creatinine have not adequately identified drugs that have generated important medical issues later in development. Here, two clinical trial databases with a posteriori known drug induced renal impairment were analyzed for the presence of a renal impairment biomarker signal from urea (590 patients; age 26-92, median 65) and creatinine (532 patients; age 26-97, median 65). Data was analyzed retrospectively using multiple definitions for the biomarker signal to include values outside stratified reference intervals, values exceeding twofold increases from baseline, values classified by the 2009 NIAID renal toxicity table, change from baseline represented as a Z-score based on intra-individual biological variations, and an adaptive Bayesian methodology that generalizes population- with individual-based methods for evaluating a biomarker signal. The data demonstrated that the adaptive Bayesian methodology generated a prominent drug induced signal for renal impairment at the first visit after drug administration. The signal was directly related to dose and time of drug administration. All other data analysis methods produced none or significantly weaker signals than the adaptive Bayesian approach. Interestingly, serum creatinine and urea are able to detect early kidney dysfunction when the biomarker signal is personalized.

Assessment of whether laboratory results in clinical studies are abnormal relies heavily on the laboratory upper or lower reference limits. While central laboratories use a single reference interval for an entire study population, local laboratories apply their own reference intervals. Due to bias caused by method differences between local laboratories, such reference intervals are not interchangeable. We have compared the reference intervals of local laboratories after harmonizing their test results, using an analytical calibration step for routine clinical biochemistry analytes. Our results show that differences in local reference intervals could result in significantly variable criteria being used for assessment of normality versus abnormality—up to more than 30%. Significant differences were observed for most analytes used in routine safety monitoring. We conclude that local reference intervals are not useful for drug toxicity trend analysis. Instead, a single reference interval after data harmonization can be used with advantage in addition to assessment of changes in individuals versus their baseline results.

Urinary kidney injury molecule (KIM-1) is a sensitive quantitative biomarker for early detection of kidney tubular injury. The objective of the present work was to analytically validate this urinary renal injury biomarker. Duo-set reagents from R&D were used to develop the ELISA and validate the assay's linearity, intra-run precision, inter-run precision, lower limit of quantification, recovery, dilutional verification, reference range, stability, and length of run. The reference range data suggests that the healthy population falls within the assay range (59 - 2146 pg/mL) and upper limit of quantitation for this assay is 17168 pg/mL for the patient population. This is a robust assay to detect urinary levels of KIM-1, which serves as a non-invasive sensitive, reproducible, and potentially high-throughput method to detect early kidney injury in drug development studies.

OBJECTIVE: Irritable bowel syndrome (IBS) is diagnosed by the presence of a constellation of symptoms fulfilling the Manning or Rome Criteria, after exclusion of organic disease. To exclude other diagnoses that might contribute to the abdominal pain or bowel symptoms experienced by subjects with IBS, numerous screening algorithms have been advocated, incorporating lactose hydrogen breath tests, thyroid function tests, fecal ova and parasite determination, and colonic endoscopy/radiography. The utility of these tests in uncovering alternative diagnoses, other than IBS, was examined in 1452 patients. METHODS: Data were combined from two large multinational studies of IBS patients. All patients exhibited symptoms meeting the Rome criteria for IBS for at least 6 months before study entry. If prior evaluation had been > 2 yr previously, patients underwent colonic endoscopy/radiography at study entry. In addition, thyroid function tests, fecal ova and parasite determination, and a lactose hydrogen breath test were performed. RESULTS: Lactose malabsorption was diagnosed in 23% (256/1122) of patients. Colonic abnormalities were detected in 2% (7/306) of patients; in four patients, colonic inflammation (n = 3) or obstruction (n = 1) may have contributed to symptoms of abdominal pain or altered bowel habits. Abnormal thyroid-stimulating hormone levels were detected in 6% (67/1209) of patients, of whom half were hypothyroid and half were hyperthyroid. Positive fecal ova and parasite tests were noted in 2% (19/1154) of patients. CONCLUSIONS: Examination of screening tests in 1452 patients with an established history of IBS revealed an incidence of lactose malabsorption comparable to that in the general U.S. population and a low incidence of thyroid dysfunction, ova and parasite infestation, or colonic pathology. The limited detection rates, added costs, and inconvenience of these tests suggest that their routine use in the diagnostic evaluation of established IBS patients should be scrutinized.

BACKGROUND A concern with using creatinine for the identification of drug-induced renal impairment is that small changes in serum creatinine (SCr) that frequently are perceived as measurement bias or imprecision translate into important changes in the glomerular filtration rate. Important drug-generated changes in creatinine are difficult to detect because they are frequently observed within the reference interval. The design of a crossover drug protocol is an opportunity to use study participants as their own control to identify these small but important changes. METHODS Twenty individuals participating in a phase I clinical trial were evaluated for SCr changes beyond those expected for biological variation according to individual Z scores derived from an adaptive Bayesian model. After 2 screening tests, participants were administered either drug (n = 11) or placebo (n = 9) during the first dosing interval. A washout period followed, and drug was then administered to the group that initially received placebo, and vice versa (10 visits total per participant). RESULTS Although all creatinine values fell within the reference interval, 8 participants individually showed increased concentrations (Z scores >2.33). These 8 participants were confirmed at unblinding to have received the drug in the identified dosing period, with 1 exception. CONCLUSIONS The ability to identify a drug effect on an individual-participant basis in early-phase studies permits drug developers to recognize issues early in development and rapidly engage in risk–benefit analysis. These results suggest that SCr monitoring is able to detect early kidney dysfunction when individual-based reference intervals are used.

Although hemoglobin concentration measurement is among the most commonly performed blood tests, the description of global population parameters, heterogeneous factors, and within-subject variations in patients with disease remains incomplete. As absolute action values are being published in the medical literature and by government healthcare agencies, these measures are important to define patient-specific ranges of biomarkers. Here, a global clinical trial data set composed of 1,537,932 hemoglobin values from 416,374 patients and 372 clinical indications was generated over 2 years by automated analyzers in a global network of 5 laboratories. Within- and between-subject components of variance and the effect of factors age, gender, nationality, and clinical indication were determined using unbalanced multiway analysis of variance. Average within-subject variances differed significantly depending on the clinical indication (0.15–1.3 g 2 /dL 2 ) but, nevertheless, remained significantly lower than between-subject variances. The main sources of between-subject variation were clinical indication and gender, followed by age and nationality. An adaptive Bayesian approach was then used to generate patient-specific ranges of hemoglobin for drug safety and efficacy assessment in clinical trials. The same methodology can be applied to the evaluation of any biomarker signal in translational medicine.

Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log 10 HBV DNA dynamic range, were thawed and stored at −70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h) and were refrozen at −70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at ≤4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at ≤4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of ≥20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at −70 or 4°C.

A rapid sensitive method for serum caffeine analysis is reported. One hundred microliters of serum is mixed with 50 microliters of the acetonitrile/internal standard mixture, centrifuged, and 10 microliters of the supernatant is injected into the liquid chromatograph. Caffeine and internal standard are eluted at 6.09 and 7.17 minutes respectively. The procedure is linear to at least 20 micrograms/milliliter. The within run precision was 2.3% and 3.8% (N=10) respectively for mean concentrations of 7.77 and 15.73 micrograms/milliter. Between day precision was 11.0% and 8.6% respectively for mean concentrations of 7.63 and 15.55 micrograms/milliliter. Recovery of caffeine over the analytical range averaged 104%.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P -450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P -450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P -450 are not distributed uniformly throughout the rat adrenal cortex.

Summary The microbial toxin trans L ¯ ¯ ‐2‐amino‐4‐methoxy‐3‐butenoic acid and its parent vinylglycine are deaminated to alpha ketoacids by sheep liver serine threonine dehydratase. The ability of the dehydratase to deaminate the above compounds is consistent with the proposed mechanism for dehydratases. Since these compounds irreversibly inhibit pig heart aspartate aminotransferase (1), the dehydratase may act as a detoxification pathway.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.

Products, substrates, and inhibitors of the threonine dehydratase from sheep liver (EC 4.2.1.16) have been investigated by proton nuclear magnetic resonance and optical rotation. The alpha-ketobutyrates produced from L-threonine and L-allothreonine in 2H2O have been shown to incorporate a single deuterium into the beta position. The dehydratase forms R-alpha-ketobutyrate-beta-d from L-threonine and L-allothreonine. The alpha protons of the substrates, threonine and allothreonine, do not exchange in the presence of the dehydratase. In the presence of dehydratase, the competitive inhibitors L-cysteine and L-alanine undergo alpha-proton exchange. Highly purified dehydratase has been used to determine kinetic parameters for the usbstrates L-threonine, L-allothreonine, L-serine, and L-chloroalanine. L-Chloroalanine, in addition to being a substrate, inhibits the dehydratase in a manner kinetically identical with that of L-serine.

Employing immunohistochemical techniques, the distributions of NADPH-cytochrome c (P–450) reductase and of two forms of cytochrome P–450, PB–B and MC–B, were examined in the livers of untreated rats and of rats pretreated for four days with phenobarbital, 3-methylcholanthrene, or pregnenolone-16α-carboni- trile. Fluorescent and peroxidase-antiperoxidase immunohistochemical staining revealed that each enzyme exhibits a characteristic pattern of intralobular distribution which is affected in characteristic manners by different xenobiotics.

Summary The contents of cytochrome P-450 and adrenal ferredoxin were determined in the adrenals of spontaneously hypertensive rats of the Wistar-Kyoto strain and were compared with those in the adrenals of normotensive Wistar-Kyoto rats. Male rats between 10 and 12 weeks of age were employed in these studies. While the adrenal weight was only slightly decreased in the spontaneously hypertensive rats, the contents of both cytochrome P-450 and adrenal ferredoxin per 2 adrenals were found to be approximately 35% lower as compared to the normotensive controls. Subcellular fractionation of whole adrenal homogenates revealed that the observed decreases in the contents of cytochrome P-450 and adrenal ferredoxin were the result of alterations in the concentrations of these proteins in mitochondria. These observations are consistent with the suggestion that the pattern of steroidogenesis in the adrenal of the spontaneously hypertensive rat is altered.

L-Serine-threonine dehydratase (EC 4.2.1.16) from sheep liver has been obtained as a highly purified preparation as shown by ultracentrifuge studies and analytical disc gel electrophoresis. The dehydratase has a molecular weight of 98,000 +/- 10,000 and is composed of two nonidentical subunits with molecular weights of 41,000 and 47,000. The 41,000 subunit is covalently linked to the carbonyl reagent-sensitive coenzyme which has been identified as alpha-ketobutyric acid.