Amadeo J. Pesce

Reference intervals: an update.

Reference intervals serve as the basis of laboratory testing and aid the physician in differentiating between the healthy and diseased patient. Standard methods for determining the reference interval are to define and obtain a healthy population of at least 120 individuals and use nonparametric estimates of the 95% reference interval. This method is less accurate if the group size is significantly less and does not allow for exclusion of outliers. In order to overcome these limitations many authors in the current literature report reference intervals after arbitrary truncation of the data or use inappropriate parametric calculations. We argue that the use of outlier removal and robust estimators, with or without transformation to normality, address the shortcomings of the standard method and eliminate the need for employing less valid methods.To test these methods of analysis well-defined test groups are required. In a few studies physician-determined health status is provided for each subject along with commonly measured analytes. The NHANES and Fernald studies provide such groups. With such data it is possible to show the range of effects on the reference interval width by including a known non-healthy subgroup. With the NHANES data the effect ranged from negligible to a 30% increase in reference interval width. We found that use of outlier detection with the robust estimator yielded reference intervals that were closer to those of the true healthy group.Another issue is one of demographics. That is, whether or not one should derive separate reference intervals for different demographic groups, e.g., males and females. The standard mathematical test for deriving separate reference intervals is due to Harris and Boyd. Using the NHANES data we examined 33 analytes for each of three ethnic groups (separated by genders). We used the Harris and Boyd procedure and observed that it was necessary to derive separate reference intervals for approximately 30% of the comparisons. The most notable analytes were glucose and gamma GT.The methods used by most laboratories have similar precision, identical units, are linearly related (often on a 1:1 basis) and correlate well with each other. As a result the only difference is the method bias. By using the reference interval width, this bias is eliminated. We argue that the log ratio of the reference interval widths is a good estimate of the variability between groups.

Urine drug screening in the medical setting.

BACKGROUND The term drug screen is a misnomer since it implies screening for all drugs, which is not possible. Current practice is to limit the testing to the examination of serum for several drugs such as ethanol, acetaminophen, salicylate, and of urine for several specific drugs or classes of drugs. In the emergency setting the screen should be performed in less than one hour. Controversies continue to exist regarding the value of urine drug testing in the medical setting. The reasons for these include the drugs involved, the sample, the methods utilized to perform the tests, and the level of understanding of the physician using the data, all of which are closely related to the other. METHODS Current automated methods provide rapid results demanded in emergency situations, but are often designed for, or adapted from, workplace testing and are not necessarily optimized for clinical applications. Furthermore, the use of these methods without consideration of the frequency in which the drugs are found in a given area is not cost-effective. The laboratory must understand the limitations of the assays used and provide this information to the physician. Additionally, the laboratory and the physicians using the data must cooperate to determine which drugs are appropriate and necessary to measure for their institution and clinical setting. In doing so it should be remembered that for many drugs, the sample, urine, contains the end product(s) of drug metabolism, not the parent drug. Furthermore, it is necessary to understand the pharmacokinetic parameters of the drug of interest when interpreting data. Finally, while testing for some drugs may not appear cost-effective, the prevention or reduction of morbidity and mortality may offset any laboratory costs. CONCLUSIONS While the literature is replete with studies concerning new methods and a few regarding physician understanding, there are none that we could find that thoroughly, objectively, and fully addressed the issues of utility and cost-effectiveness.

CYCLOSPORINE DOSE REDUCTION BY KETOCONAZOLE ADMINISTRATION IN RENAL TRANSPLANT RECIPIENTS

Cyclosporine metabolism occurs in the liver via hepatic cytochrome P-450 microsomal enzymes. Ketoconazole, an imidazole derivative, has been shown to inhibit the cytochrome P-450 enzyme system. Thirty-six renal transplant recipients receiving cyclosporine as part of a triple immunosuppressive drug regimen were started on 200 mg/day of oral ketoconazole. The dose of cyclosporine was reduced by 70% at the start of ketoconazole; this dose reduction was based on our previous experience with concomitant cyclosporine-ketoconazole therapy. Ketoconazole was started in patients who had been on cyclosporine for between 10 days and 74 months. The mean cyclosporine dose was 420 mg/day (5.9 mg/kg/day) before starting ketoconazole and 66 mg/day (0.9 mg/kg/day) one year after the addition of ketoconazole; this represents a cyclosporine dose reduction of 84.7% (P less than 0.0001). The mean trough whole-blood cyclosporine concentrations measured by HPLC, were 130 ng/mL preketoconazole and 149 ng/mL after 1 year of combination therapy. Mean serum creatinine and BUN levels were unchanged before and during ketoconazole administration, and no changes in liver function tests were noted. Cyclosporine pharmacokinetics were performed before and after at least three weeks of ketoconazole. Hourly whole-blood samples were measured by HPLC (parent cyclosporine only) and TDX (parent + metabolites). Combination therapy resulted in decreases in the maximum blood concentration and the steady-state volume of distribution divided by the fractional absorption, and increases in mean residence time and the parent-to-parent plus metabolite ratio (calculated by dividing the HPLC by the TDX value). The addition of ketoconazole to cyclosporine-treated patients resulted in a significant inhibition of cyclosporine metabolism and decrease in the dosage. There was minimal nephrotoxicity, and only four rejection episodes occurred on combined therapy. The concomitant administration of the two drugs was well tolerated, and there was no deleterious effect on the immunosuppressive activity of cyclosporine. This drug interaction provides a significant reduction in the costs associated with organ transplantation.

Binding of protein to polystyrene in solid-phase immuno assays

Abstract The concentration and time dependence of the adsorption of protein to polystyrene tubes was studied under conditions used for the competitive radioimmunassay and enzyme-linked immunosorbent assay (ELISA) techniques. The maximal adsorption of a 0.2 ml solution of rat albumin or rabbit IgG to a 12×x 75 mm polystyrene tube was about 5μg after 24h of incubation. Choosing 24h of incubation as an end point, 7% of the observed adsorption occured at 5 min at low protein concentrations (10 ng) while at high protein concentrations (5μg), 50% of the binding took place in 5 min. Using a rabbit anti-rat albumin system the amount of antigen bound to antibody under conditions similar to that used in a competitive binding assay increased during the 24h incubation period. The rate of saturation was greater with increasing concentration. At the highest concentration of albumin used, 50% saturation occured in 30 min. When the adsorption of IgG to polystyrene was studied using the ELISA assay of goat anti-human IgG, maximal immunochemical reactivity was found to be 2.25 h and this reactivity decreased with increasing incubation time. The reaction of the enzyme antibody conjugate with the adsorbed IgG was time dependent and the color yield increased with increased time of incubation of the conjugate. The binding characteristics of the competitive and ELISA techniques are different: the ELISA technique has an optimal time during which the adsorbed IgG is immunoreactive while the competitive assay approaches maximal binding in an asymptotic manner.

The Effect Of Oral Metoclopramide On The Absorption Of Cyclosporine

This study was performed to determine the effect of coadministered oral metoclopramide on the absorption of oral cyclosporine in 14 kidney transplant patients. The study was conducted on two consecutive days. Ten patients were studied twice, and 4 patients once, giving 24 studies. The total dosage of metoclopramide was 20 mg. The day on which metoclopramide was administered was chosen randomly. Whole-blood cyclosporine levels were analyzed by high-performance liquid chromatography. Coadministration of cyclosporine with metoclopramide resulted in a significant increase in mean maximum blood concentration (567 ng/ml versus 388 ng/ml) and mean area under the blood-concentration-versus-time curve (4120 ng X hr/ml versus 3370 ng X hr/ml); and a significant decrease in mean time to reach maximum concentration. The mean increase in area under the blood-concentration-versus-time curve was 29%. No significant changes were observed in the elimination of cyclosporine when it was coadministered with metoclopramide. These observations suggest that coadministered metoclopramide increased the total absorption of cyclosporine. Metoclopramide has been shown to hasten gastric emptying; since cyclosporine is absorbed predominantly in the small intestine, coadministration of metoclopramide resulted in increased bioavailability of cyclosporine.

Micropuncture Study of Albumin Transfer in Aminonucleoside Nephrosis in the Rat

Extract: Albumin concentration in the proximal tubules of normal rats and rats with aminonucleoside nephrosis (AMN) was estimated by means of a sensitive radioimmunoassay. Nephrotic animals were studied at the onset of proteinuria and when the nephrotic syndrome was fully established. Albumin concentration in the proximal tubule of normal male rats averaged 2.5 ± 0.3 mg/100 ml, whereas in early nephrotic rats it averaged 4.9 ± 1.3 mg/100 ml and in normal sized tubules of heavily proteinuric rats, 15.7 ± 3.3 mg/100 ml. Control and early AMN animals appeared to have a relatively homogeneous population of surface convolutions, whereas the fully nephrotic rats had markedly increased heterogeneity. A population of dilated proximal tubules with an albumin concentration of 233.8 ± 35.4 mg/100 ml was found in this group. Albumin concentrations were randomly distributed throughout the proximal convolution of all three groups with no clear pattern of changing concentration along the nephron. The data demonstrate that enhanced glomerular permeability to albumin occurs in the experimental nephrotic syndrome in the rat.Speculation: Early proximal tubular albumin concentrations are increased in aminonucleoside nephrosis in the rat, which indicates that there is an increased glomerular permeability to albumin in this disease. Absolute amounts of albumin leaving the end of the proximal convolution are increased over controls. Increased albumin excretion in these animals may be accounted for, in part, by increased glomerular permeability for albumin.

Correlates of zidovudine phosphorylation with markers of HIV disease progression and drug toxicity

ObjectiveTo determine the relationships between in vivo zidovudine (ZDV) phosphorylation in cells from HIV-infected patients and markers associated with disease progression and drug toxicity. DesignA pharmacokinetic study of ZDV metabolism sponsored by the AIDS Clinical Trials Group (protocol 161). Plasma and intracellular pharmacokinetics following a 100 mg oral dose of ZDV were determined at weeks 4 and 24 of initial therapy in adult patients. Plasma concentrations and phosphorylated ZDV were determined by radioimmunoassay, and area under the concentration-time curves (AUC) were compared with clinical data collected during the pharmacokinetic study. SettingAn outpatient setting at the University of Cincinnati AIDS Treatment Center, Cincinnati, Ohio, USA. PatientsHIV-infected adults with CD4+ lymphocyte counts 200–500 × 106 cells/I with no prior history of anti-HIV therapy and no active infections requiring systemic therapy. Of 30 patients enrolled, 21 were evaluable. InterventionsNone. Main outcome measuresAUC of plasma ZDV and intracellular total phosphorylated ZDV were compared with change from baseline of the following surrogate markers: CD4+ lymphocyte count, %CD4+ lymphocytes, CD4+/CD8+ cell ratio, serum β2-microglobulin, serum neopterin, neutrophils, red cell count, and hemoglobin. ResultsNo correlations between plasma AUC and markers of therapeutic response were observed. However, significant positive correlations were observed between the AUC of total phosphorylated ZDV and changes in the %CD4+ lymphocytes and CD4+/CD8+ lymphocyte ratio; a negative correlation was observed with change in hemoglobin. Patients who responded to ZDV therapy, as measured by these variables, demonstrated significantly higher intracellular AUC (>3 pmol × h/106 cells) than those who did not (approximately 2 pmol × h/106 cells). ConclusionsThe ability of HIV-infected patients to phosphorylate ZDV correlates with changes in markers associated with drug effect and toxicity. Potential individualization of therapy through monitoring of total phosphorylated ZDV in patients therefore warrants further exploration.

Plasma coenzyme Q10 reference intervals, but not redox status, are affected by gender and race in self-reported healthy adults.

BACKGROUND Abnormal concentrations of coenzyme Q(10) have been reported in many patient groups, including certain cardiovascular, neurological, hematological, neoplastic, renal, and metabolic diseases. However, controls in these studies are often limited in number, poorly screened, and inadequately evaluated statistically. The purpose of this study is to determine the reference intervals of plasma concentrations of ubiquinone-10, ubiquinol-10, and total coenzyme Q(10) for self-reported healthy adults. METHODS Adults (n=148), who were participants in the Princeton Prevalence Follow-up Study, were identified as healthy by questionnaire. Lipid profiles, ubiquinone-10, ubiquinol-10, and total coenzyme Q(10) concentrations were measured in plasma. The method used to determine the reference intervals is a procedure incorporating outlier detection followed by robust point estimates of the appropriate quantiles. RESULTS Significant differences between males and females were present for ubiquinol-10 and total coenzyme Q(10). Blacks had significantly higher Q(10) measures than whites in all cases except for the ubiquinol-10/total Q(10) fraction. CONCLUSIONS The fraction of ubiquinol-10/total coenzyme Q(10) is a tightly regulated measure in self-reported healthy adults, and is independent of sex and racial differences. Different reference intervals for certain coenzyme Q(10) measures may need to be established based upon sex and racial characteristics.

Kinetics of Oral Tolerance: Study of Variables Affecting Tolerance Induced by Oral Administration of Antigen

The induction of antigen-specific tolerance by oral administration of hen egg albumin (OVA) was shown to be time- and dose-dependent. The IgE response was the most effectively suppressed by antigen feeding prior to or after parenteral immunization. The effect on IgG response paralleled that observed on the IgE response, but the magnitude of suppression was less pronounced. The IgA response was enhanced rather than suppressed if the interval between feeding and parenteral immunization was short or if the animals were antigen-fed after the intraperitoneal priming.

Immunoregulatory properties of antigenic fragments from bovine serum albumin

Regulation of the immune response to bovine serum albumin (BSA) by defined substructures of the protein was investigated. The fragments consisted of 41 to 307 amino acids each bearing unique serologic determinants. When administered intravenously the fragments were capable of suppressing the immune response to the entire BSA molecule. The suppression was T-cell mediated and affected the determinants that were linked. The fragments also primed for a secondary anti-BSA response when given in adjuvant; however, the ability to prime was restricted to determinants on the immunizing fragment. The data demonstrate that immune responses to a large protein molecule can be regulated by a relatively small component of its structure and that the ability of a fragment to induce help is more restricted than is its suppressive potential.