Clinical utility of monitoring tacrolimus blood concentrations in liver transplant patients.

The relationship between the dose of tacrolimus, trough tacrolimus blood concentration, and selected clinical endpoints (acute rejection, nephrotoxicity, and other toxicities) were examined in a prospective, multicenter clinical trial to validate the use of an enzyme-linked immunosorbent assay (ELISA) for monitoring whole-blood concentrations of tacrolimus in liver transplant patients. A total of 111 subjects from six transplant centers were evaluated over 12 weeks posttransplantation. In addition to trough tacrolimus blood concentrations, hematocrit, ALT, AST, GGTP, alkaline phosphatase, total bilirubin, serum creatinine, BUN, serum potassium, serum magnesium, blood glucose, and serum albumin were also measured. The relationship between trough tacrolimus blood concentrations and clinical endpoints was analyzed using both a logistic regression model and a Cox proportional hazard model. By logistic regression analysis, a statistically significant (p = 0.0465) relationship between increasing trough tacrolimus blood concentrations and decreasing risk of acute rejection was demonstrated over a 7-day time window. Nephrotoxicity and other toxicities also demonstrated statistically significant relationships with trough tacrolimus blood concentrations. The results of the Cox analysis were consistent with the logistic regression analysis. Using receiver operator characteristic curves, trough tacrolimus concentrations as measured by the ELISA method were able to differentiate the occurrence of nephrotoxicity and toxicity from nonevents. To minimize nephrotoxicity of tacrolimus, it is necessary to maintain trough blood concentrations below 15 ng/ml. This study demonstrates that the ELISA method used to measure tacrolimus blood concentrations in this study provides information of predictive value for managing the risk of nephrotoxicity, other toxicity, and rejection in liver transplant patients.

Analytical validation of the PRO-Trac II ELISA for the determination of tacrolimus (FK506) in whole blood.

BACKGROUND: The analytical validation of multiple lots of the PRO-Trac II ELISA (DiaSorin) for the determination of tacrolimus in whole blood is described. METHODS: The analytical parameters assessed included analytical sensitivity, dilution linearity, functional sensitivity, values in samples containing no tacrolimus, intra- and interassay precision, supplementation and recovery, metabolite cross-reactivity, interference studies, and method comparisons HPLC-tandem mass spectrometry (HPLC/MS/MS) and the IMx Tacrolimus II multiparticle enzyme immunoassay. Where appropriate, assessments were performed according to NCCLS guidelines. RESULTS: The mean analytical detection limit was <0.25 microg/L for all lots, whereas the functional sensitivity was 1.0 microg/L. Excellent linear correlation (r = 0.985) was observed for dilution linearity. The intraassay imprecision was <7%, and the total imprecision by ANOVA was <10%. Recovery was 109% +/- 11%. Metabolite cross-reactivity was consistent with previous reports for this antibody. No interference was observed for 35 tested drugs. Method comparison with HPLC/MS/MS showed no statistically significant differences. Samples exhibited stability through four freeze/thaw cycles and for 1 week at room temperature. CONCLUSION: These data demonstrate that the PRO-Trac II ELISA is a robust, accurate, and precise tool for the assessment and management of tacrolimus blood concentrations in transplant patients.

Could education be dangerous to your health? Pitfalls of misapplied statistics.

It is important to evaluate cardiovascular risk factors. A correlation between cholesterol levels and the duration of formal education has not been observed previously. In analyzing our data, it was found that these two variables had a significant correlation (p < 0.05). Further evaluation did not substantiate this correlation. After considering various possible reasons we concluded that the seeming correlation was caused by inappropriate statistical evaluation.

Analytical evaluation of i-STAT Portable Clinical Analyzer and use by nonlaboratory health-care professionals.

We evaluated the performance of the i-STAT Portable Clinical Analyzer, a hand-held instrument that, with its current cartridge, analyzes for electrolytes, urea nitrogen, glucose, and hematocrit in approximately 60 microL of whole blood in approximately 90 s. Accuracy, imprecision, and linearity studies were performed with aqueous controls and standards and by split-sample analysis. Intrarun imprecision (CV) ranged from 0.34% to 3.97%. Total imprecision over a 2-month period ranged from 0.42% to 4.83%, with urea nitrogen and glucose analyses generating the higher values. Patients' results from the Portable Clinical Analyzer correlated well with those obtained for whole blood or plasma by the Nova Stat Profile 5, the Beckman Synchron CX3, or the Technicon H1 Hematology Analyzer, with Sylx values < 0.2 mmol/L for potassium; < 1.5 mmol/L for sodium, glucose, and urea nitrogen; < 2.4 mmol/L for chloride, and < 2.4% for hematocrit. We also ascertained imprecision and accuracy of the system placed in a cardiothoracic intensive-care unit and operated by nurses. There were no significant differences in either the imprecision or accuracy of the system in this setting. We conclude that operator technique is not a factor in the analytical performance of the system and that it can be used by nonlaboratorians with a high degree of confidence that reliable results will be obtained.

Hormone, inorganic phosphate concentrations and enzyme activity in hemodialyzed and kidney-transplanted children.

Several serum hormone concentrations, enzyme activities, and inorganic phosphate complexes were investigated in 13 hemodialyzed children, 7 kidney-transplanted children, and in 15 healthy controls. Prior to kidney transplantation 10 of the 14 tested hormone levels of hemodialyzed children differed significantly from those of healthy controls; however, after kidney transplantation most of them normalized, only the angiotensin-converting enzyme and alkaline phosphatase activities were significantly elevated in comparison with the control group. Among the different inorganic phosphate complexes, dialysis had the least effect on the CaHPO4 complex. In the hemodialyzed group the plasma renin activities were decreased and the amylase and lipase activities were increased.

Bile secretory function of intrahepatic biliary epithelium in the rat.

To shed light on ductular fluid secretion, hepatic histology and ultrastructure, cell proliferation and phenotypes, and several aspects of biliary physiology were studied in rats with ductular cell hyperplasia induced by either biliary obstruction (0-14 days) or 1-naphthylisothiocyanate (ANIT) feeding (0-28 days). In both groups of experimental animals, bile duct hyperplasia and spontaneous bile flow and secretin-induced choleresis increased with time of treatment in a linear fashion. Measurements of [14C]mannitol biliary entry and of biliary tree volume showed that the increase in both spontaneous and secretin-stimulated bile flow originated at the proliferated biliary structures. Ultrastructural examination, [3H]thymidine incorporation, and histochemical and immunohistochemical staining for various markers demonstrated that in both hyperplastic reactions the proliferated cells were the progeny of preexisting biliary epithelial cells and retained their characteristics. These results indicate that the increased bile secretory activity associated with either biliary obstruction or ANIT intoxication reflects a quantitative change due to the proliferation of biliary epithelial cells. Thus both models of bile ductular cell hyperplasia lend themselves to assessment of the transport function of intrahepatic biliary epithelium and its contribution to normal bile formation. In the present studies, we have estimated that net ductular secretion in the normal rat accounts for 10-13% of spontaneously secreted hepatic bile.

Change of serum inorganic phosphate complexes in diabetic ketoacidosis.

The concentrations of serum inorganic phosphate complexes (HPO4(2-), H2PO4-, NaHPO4-, KHPO4-, CaHPO4 and MgHPO4) were studied during diabetic ketoacidosis. On admission to hospital the H2PO4- concentration was significantly increased, in comparison with well-controlled diabetics. After 12-24 h the concentration of all phosphate complexes decreased. At 72 h only the MgHPO4 concentration was decreased. These changes are related to acidosis, insulin therapy and tubular dysfunction.

Effect of hemodialysis on the distribution of phosphates in blood.

Phosphate retention is commonly associated with chronic renal failure. Previously we presented a technique to calculate the distribution of phosphate constituents in body fluids. We studied it in pre- and postdialysis serum of 62 patients. After hemodialysis, there was some decrease in the concentration of each constituent, with significant changes in the molar fraction of H2PO4- and CaHPO4. There is a significant negative correlation between the molecular mass of the constituent (X) and the effect of dialysis (Y): urea and H2PO4- 50%; NaHPO4-, HPO4(2-), and creatinine 41-44%; MgHPO4 39%, and CaHPO4 28%. Y = 69.7 - 0.26x, r = -0.833, p less than 0.001. This limited removal of CaHPO4 may contribute to the vascular calcification and peripheral ischemic necrosis of chronic uremic patients.

Immunoprecipitation method for CK-MB analysis re-evaluated: influence of CK-BB and macro-CK on blank activities.

We evaluated the analytical performance of the immunoprecipitation technique for quantification of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB, focusing on specimens with increased blank activities. The samples we studied had blank values that were equal to or greater than either 15 U/L or the uncorrected CK-MB value. Of 134 specimens selected, 16 and 21 contained macro CK type 1 and CK-BB, respectively. All samples containing macro CK type 1 gave negative CK-MB values, even though four contained significant quantities of CK-MB. Correcting the blank value for the fluid displacement of the second antibody suspension eliminated all but three of the negative results. However, 45 specimens, negative for CK-MB by electrophoresis, gave CK-MB values ranging from 1 to 21 U/L. Furthermore, 11 of the specimens containing macro-CK type 1 were in this group. Thus, the presence of macro-CK type 1 definitely interferes with the immunoprecipitation technique. Our results indicate the need to re-evaluate the diagnostic accuracy of the immunoprecipitation technique, especially when the un-adjusted blank activities are increased.

Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.

To establish the role of the biliary epithelium in bile formation, we studied several aspects of biliary physiology in control rats and in rats with ductular cell hyperplasia induced by a 14-d extrahepatic biliary obstruction. Under steady-state conditions, spontaneous bile flow was far greater in obstructed rats (266.6 +/- 51.9 microliters/min per kg) than in controls (85.6 +/- 10.6 microliters/min per kg), while excretion of 3-hydroxy bile acids was the same in the two groups. Infusion of 10 clinical units (CU)/kg per h secretin produced a minimal choleretic effect in controls (+3.8 +/- 1.9 microliters/min per kg) but a massive increase in bile flow in the obstructed animals (+127.8 +/- 34.9 microliters/min per kg). Secretin choleresis was associated with an increase in bicarbonate biliary concentration and with a decline in [14C]mannitol bile-to-plasma ratio, although solute biliary clearance significantly increased. Conversely, administration of taurocholate (5 mumol/min per kg) produced the same biliary effects in control rats and in rats with proliferated biliary ductules. In the obstructed animals, the biliary tree volume measured during taurocholate choleresis (67.4 +/- 15.8 microliters/g liver) was significantly greater than that determined during the increase in bile flow induced by secretin (39.5 +/- 10.4 microliters/g liver). These studies indicate that, in the rat, the proliferated bile ductules/ducts spontaneously secrete bile and are the site of secretin choleresis. Furthermore, because the proliferated cells expressed phenotypic traits of bile ductular cells, our results suggest that whereas under normal conditions the biliary ductules/ducts in the rat seem to contribute little to bile formation, secretion of water and electrolytes is a property of biliary epithelial cells.

Stability of lactate dehydrogenase at different storage temperatures.

The effect of storing human serum, cord blood serum or heparinized plasma at 25 degrees C, 4 degrees C & -20 degrees C on the activity and isoenzyme distribution of lactate dehydrogenase (LD) was studied. Cellulose acetate and agarose electrophoresis, as well as an immunochemical inhibition technique, were used for isoenzyme quantification. In contrast to previous reports, cryo-instability was found only in specimens stored at 4 degrees C. Serum specimens stored at 25 degrees C and -20 degrees C retained 74% and 87% of total activity after 45 days of storage. LD-1 was stable at all three temperatures, with a maximum loss of 10%. LD-2, LD-3, LD-4, & LD-5 were most labile at 4 degrees C. Specimens that are to be analyzed for total LD or LD isoenzymes should be stored frozen or, if necessary, at room temperature, but not in a refrigerator. Thus, separate storage of specimens for cardiac isoenzymes (LD & creatine kinase) is not necessary. This may eliminate a possible source of falsely elevated LD-1/LD-2 ratios, as well as reducing the labor factor and the corresponding cost of cardiac isoenzyme determinations.

The effect of oral calcium-loading test on the distribution of urinary phosphates in healthy and hypercalciuremic children.

The distribution of inorganic phosphate ions in urine is calculated with known stability constants, measured pH and electrolyte concentrations. The distribution of phosphate ions in the urine of healthy children is considerably different from that of hypercalciuremic children. Before and after oral calcium-loading test, the most significant difference is in the molar fraction of CaHPO4.

Choleretic effects of differently structured bile acids in the guinea pig.

The effects of 10 differently structured bile acids on bile flow and composition were studied in anesthetized, bile duct-cannulated guinea pigs. At the infusion rates of 2 and 4 mumole/min/kg, all bile acids produced choleresis. The most potent was chenodeoxycholate, which increased bile flow by an average of 31.25 microliters/mumole of bile acids excreted in bile. The weakest choleretic was tauroursodeoxycholate (11.02 mu/mumole). When the choleretic activity was plotted against bile acid hydrophobicity (high-performance liquid chromatography retention factor, obtained from the literature), linearity was observed with similarly conjugated bile acids. The order of potency was deoxycholate greater than chenodeoxycholate greater than cholate greater than ursodeoxycholate, both for the glycine and taurine conjugates, and for the unconjugated bile acids as well. Conjugation was also important, and the rank ordering for the choleretic activity (unconjugated bile acids greater than glycine-conjugates greater than taurine-conjugates) was the same as that for the hydrophobicity. When the choleretic activity was plotted against bile acid micellar aggregation number (in 0.15 M NaCl at 36 degrees C, obtained from the literature), a linear, direct relationship was observed. All bile acids produced similar effects on bile electrolyte concentrations: both bicarbonate and chloride slightly declined during choleresis, whereas bile acid concentrations increased. These studies suggest that, in the guinea pig the differing choleretic activities of differently structured bile acids are not due to their forming micelles in bile of different sizes; either the more hydrophobic bile acids form vesicles, whereas the more hydrophilic form micelles; or bile acids produce choleresis, in part or exclusively, by stimulating an additional secretory mechanism, possibly an inorganic ion pump; or both.

Calculated distribution of various inorganic phosphate constituents in body fluids, based on the principles of complex equilibria.

The distribution of inorganic phosphate ions in body fluids is calculated by equilibrium studies. Equations of partial mol fractions of each complex species are calculated with known stability constants, measured pH and electrolyte concentrations. We present these results in serum and urine in health, and in some diseases, with characteristic pH values and electrolyte concentrations. Distribution of various inorganic phosphate complexes, similar to total and ionized calcium, may be of practical importance.

A new recycling technique for human placental cotyledon perfusion: application to studies of the fetomaternal transfer of glucose, inulin, and antipyrine.

A previously described technique has been modified to permit the continuously recirculating perfusion of the separate maternal and fetal circulations of an isolated cotyledon of human placenta. Viability of the perfused cotyledons was established by measurements of oxygen consumption (average, 0.18 ml/gm/hr), glucose utilization (average, 1.0 mg/gm/hr), and lactate production (less than 0.01 mumol/gm/hr), and integrity of the placental barrier by the failure of India ink, 125I-albumin, or 35S-sulfobromophthalein to cross from fetal to maternal circulation. Clearance of 3H-inulin from the fetal circuit, 0.0059 +/- 0.0005 (SE) ml/min/gm, corresponded to 2.5% of its clearance by the adult human kidney. Clearance of 14C-antipyrine was 0.013 +/- 0.003 ml/min/gm. After introduction into the fetal circuit, the observed appearance of both inulin and antipyrine in the maternal circuit closely paralleled curves predicted by a simple mathematical model. The use of a continuously recirculating perfusion system is technically feasible, and has advantages over the single-pass technique for studying transplacental transfer of metabolites with a low efficiency of extraction.

Collection and preservation of human placental blood.

High-risk premature infants require red cell transfusions for anemia. Placental blood for autologous transfusions can be collected sterilely into citrate-phosphate-dextrose and stored at 4 degrees C. During storage for 8 days, the placental red cell content of adenosine triphosphate remained normal. The 2,3,-diphosphoglycerate concentration of cells stored 8 days declined sharply; however, the P50 value of the oxyhemoglobin dissociation curve declined to 24.4 +/- 2.40 torr. During storage, placental blood underwent an exchange of extracellular Na+ and K+, but no change in glutathione content. Hemolysis was less than 1 percent. Bacteriologic and fungal cultures remained sterile. These in vitro studies suggest that human placental blood can be collected safely and preserved effectively for autologous red cell transfusion therapy.

Subjective symptoms in workers with low-level exposure to lead.

In an attempt to identify health effects associated with low-level lead exposure, 45 cable-manufacturing workers underwent clinical examinations in a cross-sectional study. Thirteen workers were in direct contact with lead-containing stabilizers, while 31 were only indirectly exposed. The directly exposed had a higher prevalence of reported neurological and gastrointestinal symptoms than those with low or insignificant lead exposure. None of the directly exposed had blood lead levels exceeding 60 micrograms per 100 ml. The clinical symptoms correlated with blood lead and zinc protoporphyrin. However, when the data were subjected to hierarchical log-linear modeling, a partial association was found between zinc protoporphyrin and symptoms, but not between blood lead and symptoms. The data suggest that non-specific neurological and gastrointestinal symptoms may occur at relatively low blood lead and zinc protoporphyrin levels, and that measurement of zinc protoporphyrin and exploration of clinical symptoms are valuable components in lead screening programs.

Effects of diuretics on urate and calcium excretion.

Forty-nine patients with gout, many with hypertension and/or renal calculi, were given hydrochlorothiazide, furosemide, or ticrynafen. Diuresis and increased clearances of sodium (Na), potassium (K), chloride (Cl), and calcium (Ca) occurred after a single dose of hydrochlorothiazide, 100 mg, or furosemide, 40 mg, orally. There was very slight change in urate and phosphorus clearances. With prolonged use of hydrochloride or furosemide, diuresis and increased electrolyte excretion disappeared. Urate and Ca excretion fell with hydrochlorothiazide. With long-term use of furosemide, urate excretion was suppressed, but Ca excretion was sustained. Ticrynafen produced diuresis and increased clearances of Na, K, and Cl. Calcium excretion was increased after a single dose and minimally decreased after long-term use. Most striking was the severe and rather sustained uricosuria. Though ticrynafen is an effective uricosuric, natriuretic, and antihypertensive agent, its hepatotoxicity and nephrotoxicity mitigate against its clinical use.