Glucose ingestion does not lower testosterone concentrations in men on testosterone therapy.

Oral calorie intake causes an acute and transient decline in serum testosterone concentrations. It is not known whether this decline occurs in men on testosterone therapy. In this study, we evaluated the change in testosterone concentrations following oral glucose ingestion in hypogonadal men before and after treatment with testosterone therapy. This is a secondary analysis of samples previously collected from a study of hypogonadal men with type 2 diabetes who received testosterone therapy. Study participants (n = 14) ingested 75 grams of oral glucose, and blood samples were collected over 2 h. The test was repeated after 23 weeks of intramuscular testosterone therapy. The mean age and body mass index of study volunteers were 53 ± 8 years and 38 ± 7 kg/m2, respectively. Following glucose intake, testosterone concentrations fell significantly prior to testosterone therapy (week 0, p = 0.04). The nadir of testosterone concentration was at 1 h, followed by recovery to baseline by 2 h. In contrast, there was no change in testosterone concentrations at week 23. The change in serum testosterone concentrations at 60 min was significantly more at week 0 than week 23 (-11 ± 10% vs 0 ± 16%, p = 0.05). We conclude that oral glucose intake has no impact on testosterone concentrations in men on testosterone therapy. Endocrinology societies should consider clarifying in their recommendations that fasting testosterone concentrations are required for the diagnosis of hypogonadism, but not for monitoring testosterone therapy.

Association of Circulating Sex Hormones With Inflammation and Disease Severity in Patients With COVID-19.

Importance: Male sex is a risk factor for developing severe COVID-19 illness. It is not known whether sex hormones contribute to this predisposition. Objective: To investigate the association of concentrations of serum testosterone, estradiol, and insulinlike growth factor 1 (IGF-1, concentrations of which are regulated by sex hormone signaling) with COVID-19 severity. Design, Setting, and Participants: This prospective cohort study was conducted using serum samples collected from consecutive patients who presented from March through May 2020 to the Barnes Jewish Hospital in St Louis, Missouri, with COVID-19 (diagnosed using nasopharyngeal swabs). Exposures: Testosterone, estradiol, and IGF-1 concentrations were measured at the time of presentation (ie, day 0) and at days 3, 7, 14, and 28 after admission (if the patient remained hospitalized). Main Outcomes and Measures: Baseline hormone concentrations were compared among patients who had severe COVID-19 vs those with milder COVID-19 illness. RNA sequencing was performed on circulating mononuclear cells to understand the mechanistic association of altered circulating hormone concentrations with cellular signaling pathways. Results: Among 152 patients (90 [59.2%] men; 62 [40.8%] women; mean [SD] age, 63 [16] years), 143 patients (94.1%) were hospitalized. Among 66 men with severe COVID-19, median [interquartile range] testosterone concentrations were lower at day 0 (53 [18 to 114] ng/dL vs 151 [95 to 217] ng/dL; P = .01) and day 3 (19 [6 to 68] ng/dL vs 111 [49 to 274] ng/dL; P = .006) compared with 24 men with milder disease. Testosterone concentrations were inversely associated with concentrations of interleukin 6 (ß = -0.43; 95% CI, -0.52 to -0.17; P < .001), C-reactive protein (ß = -0.38; 95% CI, -0.78 to -0.16; P = .004), interleukin 1 receptor antagonist (ß = -0.29; 95% CI, -0.64 to -0.06; P = .02), hepatocyte growth factor (ß = -0.46; 95% CI, -0.69 to -0.25; P < .001), and interferon γ-inducible protein 10 (ß = -0.32; 95% CI, -0.62 to -0.10; P = .007). Estradiol and IGF-1 concentrations were not associated with COVID-19 severity in men. Testosterone, estradiol, and IGF-1 concentrations were similar in women with and without severe COVID-19. Gene set enrichment analysis revealed upregulated hormone signaling pathways in CD14+CD16- (ie, classical) monocytes and CD14-CD16+ (ie, nonclassical) monocytes in male patients with COVID-19 who needed intensive care unit treatment vs those who did not. Conclusions and Relevance: In this single-center cohort study of patients with COVID-19, lower testosterone concentrations during hospitalization were associated with increased disease severity and inflammation in men. Hormone signaling pathways in monocytes did not parallel serum hormone concentrations, and further investigation is required to understand their pathophysiologic association with COVID-19.

Sample Multiplexing: Increased Throughput for Quantification of Total Testosterone in Serum by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: For high-volume assays, optimizing throughput reduces test cost and turn-around time. One approach for liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays is sample multiplexing, wherein the analyte of interest is derivatized in different specimens with reagents of different molecular weight (differential mass tagging). Specimens can then be combined and simultaneously analyzed within a single injection to improve throughput. Here we developed and validated a quantitative, sample-multiplexed LC-MS/MS assay for serum total testosterone (TT) based on this approach. METHODS: For the sample-multiplexed assay, calibrators, controls, and patient specimens were first extracted separately. After mass tagging with either methoxyamine or hydroxylamine, they were combined and injected into the LC-MS/MS system. To evaluate assay performance, we determined limit of quantification (LOQ), linearity, recovery, and imprecision. A method-comparison study was also performed, comparing the new assay with the standard LC-MS/MS assay in 1574 patient specimens. RESULTS: The method was linear from 2.5 to 2000 ng/dL, with accuracies from 93% to 104% for both derivatives. An LOQ of 1.0 ng/dL was achieved. Intra-assay and total CVs across 4 quality control concentrations were less than 10%. The assay demonstrated good agreement (Deming regression, 1.03x + 6.07) with the standard LC-MS/MS assay for the patient specimens tested (TT, 3 to 4862 ng/dL). CONCLUSION: Sample multiplexing by differential mass tagging of TT increases LC-MS/MS throughput 2-fold without compromising analytical accuracy and sensitivity.

Rapid measurement of plasma acylcarnitines by liquid chromatography-tandem mass spectrometry without derivatization.

BACKGROUND: Tandem mass spectrometry (MS/MS) is being increasingly used to identify and measure acylcarnitines in blood and urine of children suspected of having fatty oxidation disorders and other inborn errors of metabolism. Rapid MS/MS analysis requires simple and efficient sample preparation. We developed a LC-MS/MS method for the online extraction of acylcarnitines in plasma without derivatization that requires only precipitation of proteins by acetonitrile followed by centrifugation, thus increasing efficiency. METHODS: An API-3000 tandem mass spectrometer (SCIEX, Toronto, Canada) equipped with electrospray ionization (ESI), TurboIon Spray source, three Shimadzu LC10AD micropumps and autosampler (Shimadzu Scientific Instruments, Columbia, MD) was used to perform the analysis. Within-day and between-day imprecision was evaluated for 10 analytes in the MRM mode using 3 levels of controls. Accuracy was determined by comparing the method with another MS/MS procedure and by recovery experiments. Sensitivity and specificity were evaluated by identifying patient samples under a wide variety of clinical conditions. RESULTS: Within-day CVs was <10% for all analytes tested and between-day CVs ranged from 4.4% to 14.2%. The method was linear in the range between 1.0 and 100 micromol/l for C2 and 0.1 and 10 micromol/l for the other acylcarnitines. The results of the comparison study yielded r values ranging between 0.948 and 0.999. Recovery ranged from 84% to 112%. The method correctly identified patients with a variety of fatty acid oxidation disorders and organic acidemias. CONCLUSIONS: Our method is a simple procedure for the analysis of acylcarnitines in plasma with minimal sample preparation. It is thus ideal in a routine clinical setting where efficient processing of clinical samples is necessary to reduce turnaround time under conditions of high-throughput.

Determination of total and free plasma carnitine concentrations on the Dade Behring Dimension RxL: integrated chemistry system.

BACKGROUND: L-carnitine is a naturally occurring quaternary ammonium compound present in all mammalian species. Its major function is to facilitate the passage of long-chain fatty acids through the mitochondrial membrane for subsequent beta-oxidation and ketone synthesis. Clinical interest in carnitine disorders relates particularly to possible deficiency states that may result in a phenotypic spectrum that includes cardiomyopathy, skeletal myopathy, hypoglycemia and hyperammonemia. The objective of this study was to develop a method on the Dade Behring Dimension RxL analyzer for measuring free and total carnitine levels in plasma. METHODS: Plasma samples were deproteinized by ultrafiltration to remove interference by endogenous thiols. Filtrates were measured directly on the RxL for free carnitine or after alkaline hydrolysis for total carnitine by an endpoint enzymatic assay that uses carnitine acetyltransferase. RESULTS: Within-run imprecision was <5% at high and low levels for both free and total carnitine while between-day imprecision was <15%. Recovery of free carnitine from spiked plasma >90%. The method was linear between 5.0 and 150.0 micromol/l and the limit of quantification was 5.0 micromol/l. Comparison of our method with another automated procedure developed on the Hitachi 917 system using Deming regression analysis resulted in the following equations: Dimension=1.034(Hitachi)-7.44 for total carnitine (r=0.955) and Dimension=0.805(Hitachi)+1.96 for free carnitine (r=0.951), respectively. CONCLUSIONS: Our method is suitable for analyzer platforms where the level of imprecision is lower and the throughput is higher than manual methods. It also avoids the use of radioisotopes and is appropriate in labs where access to reference methods such as tandem mass spectrometry and HPLC is limited or unavailable.

Improved method for concurrent quantification of antiretrovirals by liquid chromatography-tandem mass spectrometry.

Therapeutic drug monitoring (TDM) is becoming more widespread to optimize the treatment of patients with HIV/AIDS. The analytic component of TDM requires a drug assay with high specificity, small sample volume requirements, reasonable cost, and rapid turnaround time. This study modified a procedure for the concurrent measurement of 15 antiretrovirals by tandem mass spectrometry. The upper limit of the calibration curves was extended to 10,000 ng/mL, and the matrix for standards was changed from methanol to serum. Also, an additional drug, tenofovir, a nucleotide reverse transcription inhibitor, was included in the revised/improved method. Calibration curves showed good linearity between a concentration range of 100 and 10,000 ng/mL (r > 0.997 for all drugs). Accuracy was assessed by correlation of the calibrators with proficiency testing samples spiked with known drug concentrations and yielded results within 8% of the target values.

Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges.

BACKGROUND: The Dade Dimension RxL was introduced into our laboratory at the Children's National Medical Center in an effort to increase the overall efficiency and decrease costs through consolidation of existing procedures. The objectives of this study were to evaluate the performance of the Dade Dimension RxL in a Pediatric Hospital and to establish pediatric reference ranges for this analyzer. METHODS: Within-run and between-day imprecision was evaluated for >60 analytes using two levels of controls. Method comparison studies by regression analysis were performed on eight platforms that included: Vitros 500, Bayer Immuno I, Behring Nephelometer 100, Abbott TDx, Labconco chloridometer, Bayer 2000, Cobas Mira and Hitachi 747. Pediatric reference ranges were determined by utilizing the regression equations to transform the previously established pediatric reference ranges (which employed 1000-10,000 measurements/analyte) using alternate methods to their corresponding values on the Dimension system. RESULTS: The CVs for within-run and between-day imprecision for all analytes were <10%. The overall correlation (r) of the analytes tested ranged between 0.91 and 0.99. Pediatric reference ranges were obtained for over 40 analytes. CONCLUSIONS: The Dimension RxL provides precise and accurate measurement for a wide variety of analytes including routine chemistries, drugs and hormones. Since a good correlation was observed with the alternate platforms, consolidation of these various workstations into one unit would contribute to decreased turnaround time and greatly improved staff efficiency.

IMx tacrolimus II assay: is it reliable at low blood concentrations? A comparison with tandem MS/MS.

OBJECTIVES: To compare tacrolimus concentrations in blood samples using the MEIA II and MS methods at the lower and higher ends of the clinically relevant range. METHODS: MEIA II was compared to our tandem MS/MS procedure by regression and difference plot analysis. Data from recently published CAP surveys comparing MEIA II with MS procedures are also analyzed. RESULTS: Comparison of MEIA II with our tandem MS/MS procedure by regression analysis yielded r values of 0.612 and 0.829 for tacrolimus concentrations below and above 9.0 ng/mL respectively, as determined by MEIA II. Below 9 ng/mL between day imprecision of tacrolimus controls gave CVs of 12.16 for MEIA II and 7.82 for tandem MS/MS. Addition of known amounts of tacrolimus to EDTA whole blood gave percent target values of 148 and 130 at concentrations of 5.0 and 17.5 ng/mL respectively as determined by MEIA II. Difference plots demonstrated variability in the mean bias for MEIA II of 43% and 36% at the lower and higher concentration ranges respectively. Analysis of the CAP surveys suggested that the relative positive bias and inter-laboratory variability with MEIA II was more pronounced when the MEIA II median value was below 9.0 ng/mL. CONCLUSIONS: Our results along with the CAP survey data suggest that tacrolimus concentrations below 9.0 ng/mL measured by MEIA II are questionable and should be interpreted with caution.