Conditions associated with extreme hyperferritinaemia (>3000 µg/L) in adults.

BACKGROUND: The clinical significance of extreme hyperferritinaemia has come under scrutiny with the increasing recognition of haemophagocytic lymphohistiocytosis (HLH) in adults. Most studies of hyperferritinaemia have focused on serum ferritin greater than 1000 µg/L, often in ambulatory patients. The conditions associated with more extreme hyperferritinaemia are poorly understood. AIMS: To examine conditions associated with extreme hyperferritinaemia greater than 3000 µg/L in acutely ill adults at a quaternary care hospital. METHODS: Patients with serum ferritin greater than 3000 µg/L at Vancouver General Hospital between 1 August 2011 and 1 August 2012 were identified. Those over 18 years of age and with clinical data available were included in the study. RESULTS: Eighty-three subjects were identified. Twenty-one cases (25%) were due to transfusional iron overload, 16 (19%) due to liver disease and 15 (18%) due to mixed factors. Haemophagocytic lymphohistiocytosis (HLH) was diagnosed in six of 83 patients (7%) with ferritin greater than 3000 µg/L, but six of eight patients (75%) with ferritin greater than 20 000 µg/L. CONCLUSIONS: Extreme hyperferritinaemia greater than 3000 µg/L is uncommon in adult patients. The highest serum ferritin values are seen in HLH, but the differential diagnosis for serum ferritin greater than 3000 µg/L remains broad with iron overload and liver disease being the most common causes.

Progesterone therapy increases free thyroxine levels--data from a randomized placebo-controlled 12-week hot flush trial.

OBJECTIVE: Thyroid hormones and progesterone both influence core temperature, metabolism and are crucial during pregnancy. Our objective was to discover whether progesterone therapy caused changes in thyroid physiology compared with placebo. DESIGN: Post hoc analysis from a randomized (1:1) placebo-controlled 12-week trial of oral micronized progesterone (Progesterone, 300 mg/d at bedtime) for hot flushes (vasomotor symptoms, VMS) conducted in an academic medical centre. PATIENTS: Postmenopausal euthyroid, healthy (without cardiovascular diseases or risks) women, 1-11 years since last flow on no thyroid or ovarian hormone therapy with VMS participated. MEASUREMENTS: Primary outcomes were final and 12-week changes in TSH, FreeT3 and FreeT4 on progesterone vs placebo. RESULTS: Women with thyroid data (69 of 133 in original trial) were randomized to progesterone (n = 39) or placebo (n = 30)-baseline thyroid values were normal. There were no VMS-thyroid interactions-VMS Score (number × intensity) did not correlate with TSH, FreeT3 or FreeT4 (Spearman's rank correlations: -0.03 to -0.19, respectively; all P > 0.15). At 12 weeks on progesterone, TSH levels tended to be lower (1.7 mU) than on placebo (2.2), P = 0.06; FreeT4 levels were higher (16.4 pmol/l) than on placebo (15.3), P = 0.02. FreeT3 was unchanged throughout. Analysis of covariance showed a significant increase in FreeT4 on progesterone (+2.5 pmol/l; 1.9-3.0) vs on placebo (+1.7; 1.1-2.4) with 95% CI of difference = 0.8 pmol/l [0.0, 1.6], P = 0.04. CONCLUSIONS: Progesterone caused a significant FreeT4 increase that was discovered during this randomized controlled VMS trial. The clinical importance of this increased FreeT4 level remains to be documented. Registered at ClinialTrials.gov#NCT00152438.

Derivation and validation of a formula to calculate the contribution of ethanol to the osmolal gap.

STUDY OBJECTIVES: We sought to evaluate the relationship between osmolal gap and serum ethanol level and derive a formula that can be used clinically to calculate the expected osmolal gap in the presence of ethanol. Some investigators have noted that the residual osmolal gap appears to increase as the ethanol level increases, and thus it is important to determine the exact relationship between these 2 values. METHODS: In part 1, a convenience sample of emergency department patients undergoing serum ethanol determination had sodium, urea, and glucose levels and osmolality determined on the same blood sample, and values were prospectively recorded. Predicted osmolality excluding ethanol was calculated with the following formula: 2 Na (mEq/L) + (Urea [mg/dL])/2.8 + (Glucose [mg/dL])/18. The osmolal gap was determined by subtracting the calculated serum osmolality excluding ethanol from the measured serum osmolality. Linear regression analysis was then used to derive a formula for the relationship between ethanol and the osmolal gap. This formula was then prospectively validated on a second convenience sample of patients. In part 2, we repeated this experiment in vitro by adding known amounts of ethanol to serum. RESULTS: We derived the formula to calculate the contribution of ethanol to the osmolal gap by using 98 observations. The mean ethanol level was 197.8 mg/dL (SD 138.5), with a range of 0 to 538.2 mg/dL. The relationship between ethanol and osmolal gap was linear, with a Pearson coefficient of correlation of 0.99. Linear regression analysis generated a model with the following formula: Osmolal gap=(Ethanol [mg/dL])/3.7 - 0.35 or, in SI units: Osmolal gap (mOsm/kg)=1.25 (Ethanol [mmol/L]) - 0.35 The 95% confidence interval (CI) for the multiplicative factor was 1/3.58 to 1/3.80 (or, in SI units, 1.21 to 1.28). The 95% CI for the additive constant was -2.19 to 1.50. We prospectively validated our formula on 128 patients. The mean residual osmolal gap for this group of patients was 0.84 mOsm/L (SD 5.65; range, -18.40 to 17.85 mOsm/L). The results of the in vitro experiments were similar. CONCLUSION: Our data suggest that the best formula for the calculation of the contribution of ethanol to osmolality is as follows: Ethanol (mg/dL)/3.7 or, in SI units: 1.25 (Ethanol [mmol/L])

Salivary duct carcinoma secreting prostate-specific antigen.

Prostate-specific antigen (PSA) is a 30 kDa glycoprotein serine protease that shows high tissue specificity for prostatic tissue, both benign and malignant. However, recent reports have shown that a variety of normal and neoplastic tissue types express PSA immunohistochemically. In addition, rare instances of the secretion of PSA by nonprostatic cancers have been reported in the literature. The authors present a case of salivary duct carcinoma associated with elevated serum levels of PSA. Both the primary tumor and metastases stained positively with anti-PSA monoclonal antibodies, but were negative with antibodies directed against prostate-specific acid phosphatase. Elevated serum PSA levels were confirmed with three different immunoassay methods. A peak serum level of 140 micrograms/L was measured and this correlates with levels of PSA associated with metastatic prostatic carcinoma. High performance liquid chromatography with a molecular sieve column characterized the serum PSA into both free protein (approximately 20%) and protein bound to alpha-1-antichymotrypsin (PSA-ACT)(approximately 80%). Molecular weights of the free PSA and PSA-ACT subfractions were 27-31 kDa and 100-110 kDa, respectively.

Indocyanine green clearance as a predictor of successful hepatic resection in cirrhotic patients.

Twenty-two cirrhotic patients who underwent hepatic resection from July 1989 to March 1991 at Vancouver General Hospital were analyzed prospectively in order to determine whether there was any preoperative evaluation of liver function that would help identify those patients who would not survive hepatic resection. Patients were analyzed on the basis of age, type of resection, and a variety of so-called standard liver function tests. In addition, all patients were evaluated preoperatively with an indocyanine green (ICG) clearance test, a relatively new test that we attempted to evaluate in terms of its role in hepatic resection in cirrhotic patients. Parametric statistical evaluation used included Student's t-test and multivariate regression, as well as discriminate analysis. The nonparametric evaluation used was the Wilcoxon rank sum test. Overall, the 30-day mortality rate was 18%, with those patients who did not survive resection having a significantly lower ICG clearance than those who underwent successful resection (p less than 0.0001). No other liver function test was useful in determining the outcome of resection. Similarly, neither age nor type of resection appeared to influence outcome. Use of discriminate analysis (p = 0.0029) allowed the identification of a cutoff point for ICG clearance below which hepatic resection should not be attempted.

Quantitative fluorometric screening test for fecal porphyrins.

We describe a fluorometric method for screening and quantifying porphyrins in stool. A small sample of stool is extracted with concentrated HCI and is diluted 200-fold in 3 mol/L HCI before analysis. An excitation scan is done from 350 to 450 nm, monitoring emission at 603 nm. Total porphyrin is estimated at the isosbestic point for coproporphyrin and protoporphyrin (402.5 nm). Monitoring emission at 603 nm eliminates interference from chlorophyll, obviating the need for extraction with ether. The position of the excitation peak gives some indication of the nature of the porphyrins in the stool. The acid extract can be injected directly into an HPLC system for fractionation studies. Our method correlates well with the spectrophotometric method developed by Lockwood et al. (Clin Chem 1985;31:1163-7). However, in our method, the sample is easier to process and the assay has higher sensitivity than their assay. The reference interval for porphyrin in healthy individuals by the fluorometric method is less than 300 nmol/g dry weight. We can detect as little as 1 nmol of porphyrin per gram (dry weight) of stool. Results of the method vary linearly with stool porphyrin concentrations as great as 4000 nmol/g dry weight. The within-run imprecision of the method is 3%.

Low concentration galactose determination in plasma adapted to the Cobas-Bio.

Galactose elimination at blood concentrations lower than 2.22 mmol/L has been advocated as a measure of functional liver blood flow. We have adapted an assay employing galactose dehydrogenase (EC 1.1.1.48) to the Cobas-Bio to measure low galactose concentrations in plasma. The collection of blood in sodium fluoride/potassium oxalate anticoagulant tubes eliminated the necessity for the plasma deproteinization step required in similar, manual methods. The between run CV's for plasma samples spiked with galactose to concentrations of 0.13-0.5 mmol/L were 3.6% and 3.2%, respectively. Our automated assay was more precise and had a greater range of linearity than a manual galactose oxidase (EC 1.1.3.9) method set up in our laboratory (0.04-1.10 mmol/L as compared to 0.06-0.56 mmol/L). The total assay time was 20 min.

Screening tests for porphobilinogen are insensitive. The problem and its solution.

Standard screening tests for porphobilinogen (PBG) do not turn positive until the concentration of PBG exceeds 10-20 times the upper limit of normal. The authors have developed a screening procedure that uses a new ion-exchange resin to separate PBG from interfering substances in urine. On addition of Ehrlich's reagent, the color is more intense than that produced in the Watson-Schwartz test, and its spectrum more closely resembles that of pure PBG. By measuring the absorbance of this solution at 555 nm, it is possible to discriminate between urine samples with 9 mumol/L (2 mg/L) of PBG and those with no detectable PBG. The new screening test had positive results in two patients with latent acute intermittent porphyria; the Watson-Schwartz test had negative results in both cases. This procedure is easy to perform, has much greater sensitivity than the Watson-Schwartz test, and uses objective spectrophotometric data to separate positive from negative results.

Liquid-chromatographic assay of urinary porphobilinogen.

This is a rapid (10 min per sample), highly sensitive procedure for quantifying urinary porphobilinogen (PBG). Interfering substances are removed by selectively adsorbing PBG onto an ion-exchange resin. After PBG is eluted with 0.5 mol/L formic acid, Ehrlich's reagent is added to produce the chromophore, which is then injected into a liquid chromatograph equipped with a diode-array detector. PBG is separated by a linear gradient (10% to 100%) of methanol in 10 mmol/L phosphate buffer, pH 3.0. Absorbance is monitored at 555 nm. Assay response varies linearly with PBG concentration over the range 0-110 mumol/L (0-25 mg/L). As little as 1.5 mumol/L (0.3 mg/L) can be detected. In prepared urine samples with known PBG concentrations, the within-run coefficient of variation (CV) ranged from 1.7% to 3.2%, the day-to-day CV from 3.5% to 6.1%. PBG concentrations in 24-h urine collected from 25 healthy subjects were all below the detection limit of the assay (less than 1.5 mumol/L). We used the new assay to measure PBG concentrations in the urine of two patients with latent porphyria. This method is more sensitive than spectrophotometric techniques currently used for measuring urinary PBG.

Digoxin-like immunoreactivity, displacement of ouabain and inhibition of Na+/K+ ATPase by four steroids known to be increased in essential hypertension.

An endogenous digoxin-like immunoreactive substance(s) (DLIS, "endoxin") may be of significance in the etiology of essential hypertension (EH). Progesterone, dehydroepiandrosterone sulphate (DHEA-S), 11-deoxycortisol and 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), four steroids known to be increased in essential hypertension, were found to have digoxin-like immunoreactivity at levels 1,000 times higher than physiological concentrations. Of these steroids, progesterone and 18-OH-DOC were the most efficient in displacing 3H-ouabain from canine kidney Na+/K+ ATPase whereas progesterone and 11-deoxycortisol were the most potent inhibitors of this enzyme's activity. Although 18-OH-DOC and DHEA-S cross-reacted with digoxin-specific antibodies, their ability to inhibit Na+/K+ ATPase activity was minimal. Although it is concluded that these steroids may contribute to DLIS as isolated from hypertensive patients, it is unlikely that they would be of physiological significance in the etiology of EH unless they were to accumulate and act synergistically within vascular wall smooth muscle tissues.

A study into the nature and organ source of digoxin-like immunoreactive substance(s) in the perinatal period.

Digoxin-like immunoreactive substance(s) (DLIS) was isolated from sera and autopsy-derived tissue obtained from premature and full-term neonates. The highest tissue level of DLIS was in the small bowel followed by the adrenal, gallbladder and liver. Of the fluids examined, meconium had the highest level of DLIS. Preparative high performance liquid chromatography fractionation of cord blood generated at least six different fractions which not only contained DLIS material but also inhibited canine kidney Na+/K+-ATPase activity. Recovery/inhibition studies indicated that 72% of the canine kidney Na+/K+-ATPase inhibition within one fraction could be accounted for on the basis of progesterone content of the fraction.

A rapid and accurate spectrofluorometric method for quantification and screening of urinary porphyrins.

We describe a fluorescence method for screening and quantifying urinary porphyrins. New and effective approaches are used to oxidize prophyrinogens, correct the baseline, and ensure that uroporphyrin (uro) and coproporphyrin (copro) are equally detected, mole for mole. No preliminary purification is required. A 45-microL aliquot of urine is oxidized with 3 mmol/L iodine in 3 mol/L HCl to convert porphyrinogens to porphyrins, and then decolorized with 5 mL of 0.45 mmol/L sodium thiosulfate. An excitation scan is done from 350 nm to 440 nm, monitoring emission at 650 nm. Total porphyrin content is determined at the isosbestic point for uro and copro, and the mole fractions of uro and copro are estimated from the wavelength of the signal maximum. There is no interference from protein, glucose, bilirubin, or hemoglobin in high concentration. The limit of detection is less than 30 nmol/L and linearity is maintained up to 3200 nmol/L. Recoveries and precision are excellent. This is a rapid, sensitive screen for porphyrinuria as well as an accurate and precise quantitative method. We compared the method with existing methods and discuss some shortcomings common to many of them.

Minimizing analytical interferences from digoxin-like immunoreactive substances (DLIS) in cases of digoxin toxicity.

Recently, the value of therapeutic drug monitoring for digoxin has been called into question by the finding of endogenous digoxin-like immunoreactive substances (DLIS) in the serum of individuals, especially premature and full-term neonates, not being treated with digoxin. In some cases, values have been as high as 10 micrograms/L. Levels as high as 20 micrograms/L and 80 micrograms/g can be found in bile and meconium. Because of the magnitude of this interference, it is essential that methods be developed for measuring digoxin in the presence of DLIS. This is particularly important when such analyses are required in forensic science cases of suspected digoxin toxicity. This report outlines the high performance liquid chromatographic (HPLC) and radioimmunoassay (RIA) methods that we used in assessing the relative contribution made by digoxin, its metabolites, and DLIS to serum and tissue digoxin concentrations obtained by RIA in a forensic pediatric case of suspected digoxin toxicity.