Lindsay A. L. Bazydlo

The Molecular Biology of Human Iron Metabolism

Iron is one of the most important nonorganic substances that make life possible. Iron plays major roles in oxygen transport (eg, hemoglobin; -67% of total body iron [TBI]), short-term oxygen storage (eg, myoglobin; -3.5% of TBI), and energy generation (eg, cytochromes; -3% of TBI). Iron also serves vital roles in various nonheme-containing enzymes (-2% of TBI). Figure 1 lists heme-containing and nonheme iron-containing proteins. TBI is controlled by the rate of iron absorption; there are no physiologic mechanisms to excrete excess iron. Iron deficiency has many adverse consequences, including anemia, and in children, behavioral and learning disorders. Iron excess is toxic to the body, harming the heart, liver, skin, pancreatic islet beta cells, bones, joints, and pituitary gland. Maintaining proper iron balance is essential for maintaining homeostasis and health. TBI in adults normally ranges between 3.5 and 5.0 g. A total of 75% of TBI is functional, and 25% is stored within cells as ferritin or hemosiderin. Ferritin contains 24 subunits of light chains (L chains; 19.7 kDa) and heavy chains (H chains; 21.1 kDa). The L chains are encoded on chromosome 19q13.33 and are 175 amino acids long. The H chains are encoded on chromosome 11q1 and are 183 amino acids long. Each ferritin molecule can contain as many as approximately 4500 ferric ions. Because the major role of iron is in hemoglobin synthesis, this review will focus on iron, iron transport, and hematopoiesis.

New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, enabling label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without prior DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques.

Urine Spiking in a Pain Medicine Clinic: An Attempt to Simulate Adherence

Dear Editor: Urine drug test methods are classified as either screening or confirmatory. Immunoassay-based point-of-care (POC) drug screens offer results within minutes, often allowing physicians to make real-time clinical decisions ⇓. Immunoassay also has important limitations, including limited test menus; inability to distinguish among members of a drug class; inability to differentiate drugs from their metabolites; inability to quantitate compounds; and cross-reactivity with nontargeted compounds. For confirmatory analysis, mass spectrometry in conjunction with liquid or gas chromatography is commonly utilized to supplement immunoassay. This analytical technique, while more expensive and time-consuming than immunoassay, allows highly sensitive and specific identification of analytes by measuring the distinctive mass-to-charge ratios of ions. However, there is a lack of general agreement on the indications for confirmation testing. Another critical consideration is tampered urine samples. To conceal inappropriate drug use, individuals may substitute with drug-free synthetic or authentic urine, or add oxidizing chemicals or other adulterates to the samples that could interference with the test performance ⇓. Such donor behavior sometimes necessitates that toxicology laboratories perform specimen validity tests in addition to drug analysis. Buprenorphine/naloxone is U.S. Food and Drug Administration-approved for the treatment of opioid dependence. It is also used off-label for analgesia, particularly for patients with co-occurring chronic pain and opioid …

Quantitation of 25-hydroxyvitamin D2 and D3 in serum and plasma by LCMS/MS

OBJECTIVE We developed and validated an analytical method for quantifying 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in serum and plasma. METHODS Samples, pretreated with zinc sulfate and methanol, were extracted with hexane. Separation was achieved via UHPLC and 25OHD quantification was accomplished by a triple quadrupole mass spectrometer. RESULTS Imprecision was 3.6-15.1%CV and bias 88.0-126.0%. Extraction efficiency was 76.5-110.5%, whereas the matrix effect ranged from -46.7 to -32.0%. The method was applied to authentic specimens. The results showed no significant difference between serum and plasma; strong correlation with paired values from an external laboratory; and analyte stability for 15 days. CONCLUSION This method provides reliable and accurate measurement of 25OHD for use in clinical practice.

Prevalence of Rare Hemoglobin Variants Identified During Measurements of Hb A1c by Capillary Electrophoresis

To the Editor: Hemoglobin (Hb)1 variants represent a challenge to the validity of measurements of Hb A1c. Hb variants can falsely increase or decrease reported Hb A1c results, depending on the measurement procedure used (1, 2). Moreover, Hb A1c results in patients who harbor an Hb variant can be clinically misleading if the variant alters the lifespan of erythrocytes. The effects of common hemoglobin variants (Hbs C, D, E, F, and S) on some measurement procedures for Hb A1c have been documented (2, 3), including a recent study in Clinical Chemistry (4). More than 1000 other, rarer hemoglobin variants have been identified, however, and little is known about the effects of most of these variants on measurement procedures …

Steps to diagnosis of a case of surreptitious intake of one of the newer direct oral anticoagulants: a case report and literature review.

Little is known about the effects of newer oral anticoagulants on various coagulation factors. When presented with a case of intentional or suspected overdose with an abnormal coagulation profile, it is imperative to have a working diagnostic algorithm to narrow the cause to a specific drug or drug class. This may become more crucial and time sensitive when dealing with a case of acute hemorrhage. Here we discuss the first reported case of what appears to be a surreptitious intake of newer oral anticoagulants and the steps leading to the diagnosis.

Calcium, Magnesium, and Phosphate

### Calcium #### Overview of Analyte Calcium plays significant roles in cell structural components and biochemical functioning through signaling within and external to the cell. Calcium is essential for normal neuromuscular function and for correct functioning of the coagulation factors. It is the most prevalent cation in the body and is found in the skeleton, soft tissue, and extracellular fluid. In serum, the total calcium concentration is approximately 8.4 to 10.2 mg/dL (2.1–2.6 mmol/L). This calcium exists in 2 different forms, namely, bound and free. The protein-bound form of calcium accounts for approximately 40% of total serum calcium, of which 80% is bound to albumin and the remaining 20% is bound to globulins.1 A total of 10% of the total calcium is bound to small anions, including bicarbonate, phosphate, citrate, and lactate.1 The free, physiologically active calcium (often called ionized calcium) concentration in the plasma is approximately 4.4 to 5.2 mg/dL (1.1 to 1.3 mmol/L). The intracellular cytoplasmic calcium concentration is typically very low in unstimulated cells—approximately 0.1 μM. This is 4 orders of magnitude lower than the serum free-calcium concentration. The cellular machinery is exquisitely responsive to very small elevations in cytoplasmic calcium, making it a highly sensitive second messenger. The amount of serum calcium that is bound to plasma protein can be impacted by a number of factors, one of which is the pH of blood. In a patient with alkalosis, the higher pH allows for greater binding of free calcium to proteins, effectively decreasing the concentration of free calcium. Ionized calcium will decrease 0.16 mg/dL (0.04 mmol/L) with every 0.1-unit increase in pH in patients with acute respiratory alkalosis.2 Scientists have developed various empirical formulae to adjust the total serum calcium measurement according to the serum protein concentration. This method is used to exclude protein effects when interpreting total calcium … [↵][1]* To whom correspondence should be addressed. E-mail: lbazydlo{at}ufl.edu [1]: #xref-corresp-1-1

Recognition of rare hemoglobin variants by hemoglobin A 1c measurement procedures

BACKGROUND Unrecognized hemoglobinopathies can lead to measured hemoglobin A1c (Hb A1c) concentrations that are erroneous or misleading. We determined the effects of rare hemoglobin variants on capillary electrophoresis (CE) and HPLC methods for measurement of Hb A1c. METHODS We prospectively investigated samples in which Hb A1c was measured by CE during a 14-month period. For samples in which the electropherograms suggested the presence of rare hemoglobinopathies, hemoglobin variants were identified by molecular analysis or by comparison with electropherograms of known variants. When sample volume permitted, Hb A1c was measured by 2 HPLC measurement procedures and by boronate affinity HPLC. RESULTS Hb A1c was measured by CE in 33,859 samples from 26,850 patients. 15 patients (0.06%) were identified as having rare hemoglobinopathies: Hbs A2 prime, Agenogi, Fannin-Lubbock I, G Philadelphia, G San Jose, J Baltimore, La Desirade, N Baltimore, Nouakchott, and Roanne. Among 6 of these samples tested by 2 ion-exchange HPLC methods, the rare Hb was detected by both HPLC methods in only one sample, and none were detected by boronate affinity HPLC. The mean of the Hb A1c results of 2 HPLC methods differed from the result of the CE method by 0.7-2.2% Hb A1c in samples with variant hemoglobins versus <0.2% Hb A1c in samples without variants. CONCLUSION Measurement procedures differ in the ability to detect the presence of rare Hb variants and to quantify Hb A1c in patients who harbor such variants.

The sample that would not clot

BACKGROUND Vitamin K is a vital component within both the intrinsic and extrinsic coagulation cascade as certain factors (II, VII, IX, X and protein C and S) utilize vitamin K as a cofactor during post translational modification. Deficiency of vitamin K can result in the inability to properly form blood clots, both in vivo and in vitro, due to reduced vitamin K dependent factor levels and function. Vitamin K deficiency can result from congenital causes, such as VKOR or CYP2C9 mutations, or acquired causes, such as nutritional deficiencies, antibiotic therapy, or supra-therapeutic warfarin dosing. RESULTS In this case we present a patient with multifactorial vitamin K deficiency (due to nutritional defects and multiple genetic mutations in VKOR and CYP2C9) that was exacerbated by antibiotic and warfarin therapy during her hospital admission. CONCLUSION This case displays the importance of genetic testing prior to warfarin dosing and the role antibiotics play in the coagulation cascade.

Concerning the Sixth Edition of Garriott's Medicolegal Aspects of Alcohol

To Randall Baselt’s excellent review of Garriott’s Medicolegal Aspects of Alcohol, 6th edition, wewish to provide an addendum. We would like to acknowledge an essential misunderstanding between the Editors and Professor A.W. Jones, the author of chapters 3 and 4 in the 5th edition of Garriott’s Medicolegal Aspects of Alcohol. Acting on our understanding that it was Professor Jones’ wish not to revise his chapters for the 6th edition, and while exercising traditional publishers’ copyright and editorial prerogatives, we believed that we were respecting his wishes by assigning his two chapters from the 5th edition to new authors. Chapter 3 in the 6th edition, entitled ‘Disposition and Fate of Ethanol in the Body’ with R.B. Forney, Jr. as the author, was virtually identical to Professor Jones’ chapter 3 from the 5th edition of the book. This comprehensive treatise included the chapter outline, the entire text, 44 original figures, 17 tables of data and more than 600 literature references. Dr. Forney, after review and validation of Professor Jones’ work, only added 9 historical citations to the list of references, papers by R.B. Forney, Sr. Chapter 4 in the 6th edition, entitled ‘Biomarkers of Acute and Chronic Alcohol Ingestion’, which was specifically lauded by Dr. Baselt in his review, was a revised and updated version prepared by Drs. Roger Bertholf and Lindsay Bazydlo. The outline, much of the text, figures, tables and references in the original chapter by Professor Jones, remained unchanged in the 6th edition. Updates included the discussion of glucuronide and sulfate metabolites of ethanol measured in urine to detect ethanol use within the previous several days, and a broader discussion of statistical parameters applied to laboratory methods used to detect drug use. But in large part, the extensively referenced chapter 4 represented the exceptional work of Professor Jones. Although Professor Jones’s authorship of Chapters 3 and 4 in the 5th edition is acknowledged as a footnote on the title page of the 6th edition chapters, this attribution did not provide sufficient credit to his original work. We deeply regret any discourtesy to Professor Jones that may have occurred as a result of our oversight.