Assessment of Erythrocyte Transketolase, Whole Blood Thiamine Diphosphate, and Human Milk Thiamine Concentrations to Identify Infants and Young Children Responding Favorably to Therapeutic Thiamine Administration: Findings from the Lao Thiamine Study, a Prospective Cohort Study.

Background: There is limited information on relationships among biomarkers of thiamine status (whole blood thiamine diphosphate [ThDP], erythrocyte transketolase activity coefficient [ETKac], and human milk thiamine [MTh]) and clinical manifestations of thiamine deficiency. Objectives: This study aimed to explore correlations among these biomarkers and thiamine responsive disorders (TRDs), a diagnosis based on favorable clinical response to thiamine. Methods: Hospitalized infants and young children (aged 21 d to <18 mo) with respiratory, cardiac, and/or neurological symptoms suggestive of thiamine deficiency were treated with parenteral thiamine (100 mg daily) for ≥3 d alongside other treatments and re-examined systematically. Clinical case reports were reviewed by 3 pediatricians, who determined TRD or non-TRD status. Children in a community comparison group were matched by age, sex, and residence. Venous whole blood ThDP and MTh were determined by high-performance liquid chromatography fluorescence detection and ETKac in washed erythrocytes by ultraviolet spectrophotometry. Associations between biomarkers were assessed using Spearman correlations, and biomarker cutoffs predictive of TRD and ETKac >1.25 were explored using area under the receiver operating characteristic curve framework. Results: Thiamine biomarkers were available for 287 hospitalized children and 228 community children (mean age 4.7 mo; 59.4% male). Median (interquartile range [IQR]) ThDP and ETKac were 66.9 nmol/L (IQR: 41.4, 96.9 nmol/L) and 1.25 nmol/L (IQR: 1.11, 1.48 nmol/L), respectively, among hospitalized children, and 64.1 nmol/L (IQR: 50.0, 85.3 nmol/L) and 1.22 nmol/L (IQR: 1.12, 1.37 nmol/L) among 228 community children (P > 0.05 for both). Forty-five percent of breastfeeding mothers of infants <6 mo had MTh <90 µg/L. ThDP and ETKac, but not MTh, were significantly different between 152 children with TRD and 122 without TRD, but overlapping distributions undermined prediction of individual responses to thiamine. Conclusions: Although ETKac, ThDP, and MTh are useful biomarkers of population thiamine status, none of the biomarkers reliably identified individual children with TRD. ThDP is more practical for population assessment because preparing washed erythrocytes is not required.This trial was registered at clinicaltrials.gov as NCT03626337.

Protocol for measuring erythrocyte glutathione reductase activity coefficient to assess riboflavin status.

Riboflavin (vitamin B2) is a component of the co-enzyme flavin adenine dinucleotide (FAD). The activity coefficient of erythrocyte glutathione reductase (EGRAC), a FAD-dependent enzyme, is a biomarker of riboflavin status. Here, we describe a protocol for measuring unstimulated (basal) and FAD-stimulated (activated) erythrocyte glutathione reductase activity to calculate EGRAC. We describe the steps for preparing washed red blood cells and hemolysates; preparing reagents; loading, incubating, and reading the 96-well plate; and calculating the results. For complete details on the use and execution of this protocol, please refer to Hess et al.1.

A randomised study of nurse collected venous blood and self-collected dried blood spots for the assessment of cardiovascular risk factors in the Understanding Society Innovation Panel.

Dried blood spot (DBS) sample collection has been suggested as a less invasive, cheaper and more convenient alternative to venepuncture, which requires trained personnel, making it a potentially viable approach for self-collection of blood on a large scale. We examine whether participants in a longitudinal survey were willing to provide a DBS sample in different interview settings, and how resulting cardiovascular risk biomarkers compared with those from venous blood to calculate clinical risk. Participants of the Understanding Society Innovation Panel, a representative sample of UK households, were randomly assigned to three modes of interview. Most participants (84%) were interviewed in their allocated mode. Participants (n = 2162) were interviewed by a nurse who collected both a blood sample by venepuncture and a DBS card ('nurse collection') or participants were seen by an interviewer or took part in the survey online to self-collect a DBS card ('self-collection'). All DBS cards were returned in the post after the sample had dried. Lipids (total cholesterol, HDL-cholesterol, triglycerides), HbA1c and C-reactive protein were measured in venous and DBS samples and equivalence was calculated. The resultant values were used to confirm equivalent prevalence of risk of cardiovascular disease in each type of blood sample by mode of participation. Of participants interviewed by a nurse 69% consented to venous blood sample and 74% to a DBS sample, while in the self-collection modes, 35% consented to DBS collection. Demographic characteristics of participants in self-collection mode was not different to those in nurse collection mode. The percentage of participants with clinically raised biomarkers did not significantly differ between type of blood collection (for example, 62% had high cholesterol (> 5 mmol/l) measured by venepuncture and 67% had high cholesterol within the self-collected DBS sample (p = 0.13)). While self-collected DBS sampling had a lower response rate to DBS collected by a nurse, participation did not vary by key demographic characteristics. This study demonstrates that DBS collection is a feasible method of sample collection that can provide acceptable measures of clinically relevant biomarkers, enabling the calculation of population levels of cardiovascular disease risk.

Protocol and application of basal erythrocyte transketolase activity to improve assessment of thiamine status.

Thiamine (vitamin B1) is an essential micronutrient required as a cofactor in many metabolic processes. Clinical symptoms of thiamine deficiency are poorly defined, hence biomarkers of thiamine status are important. The erythrocyte transketolase activity coefficient (ETKac) is a sensitive measure of thiamine status, but its interpretation may be confounded where the availability of the transketolase enzyme is limited. Basal ETK activity per gram of hemoglobin provides a complementary biomarker of thiamine status; however, its measurement and calculation are poorly described. Here, we describe in detail the assessment of basal ETK activity, including the calculation of path length in microplates and the molar absorption coefficient of NADH specific to the assay, and the measurement of hemoglobin in sample hemolysates. To illustrate the application of the methods, we present ETKac and basal ETK activity from women in The Gambia and UK. In conclusion, we present a clear protocol for the measurement of basal ETK activity that will permit the harmonization of methods to improve replication between laboratories.

Towards harmonization of directly measured free 25-hydroxyvitamin D using an enzyme-linked immunosorbent assay.

The majority of circulating 25-hydroxyvitamin D (25(OH)D) is protein bound and perhaps less available than the free fraction of 25(OH)D; therefore, researchers have proposed that the measurement of free 25(OH)D in human serum may be a better indicator of vitamin D health status than total 25(OH)D. The availability of a new enzyme-linked immunosorbent assay (ELISA) for the determination of free 25(OH)D provides a method for direct measurement of the low levels of non-protein bound 25(OH)D. As an initial step towards harmonization of measurements of free 25(OH)D, the ELISA was used to measure free 25(OH)D in three existing Standard Reference Materials (SRMs): SRM 972a Vitamin D Metabolites in Frozen Human Serum, SRM 2973 Vitamin D Metabolites in Frozen Human Serum (High Level), and SRM 1949 Frozen Prenatal Human Serum. Target values for free 25(OH)D in the nine SRM serum pools, obtained by combining the results from two laboratories, ranged from 3.76 ± 0.36 to 10.0 ± 0.58 pg/mL. Of particular significance is the assignment of free 25(OH)D target values to SRM 1949, which consists of four serum pools from non-pregnant female donors of reproductive age and pregnant women in each of the three trimesters and which also has values assigned for vitamin D binding protein, which increases during pregnancy. The availability of target values for free 25(OH)D in these SRMs will allow researchers to validate new analytical methods and to compare their results with other researchers as an initial step towards harmonization of measurements among different studies and laboratories.

Delayed Processing of Chilled Whole Blood for 24 Hours Does Not Affect the Concentration of the Majority of Micronutrient Status Biomarkers.

BACKGROUND: The measurement of micronutrient status is essential to understand the health of individuals and populations, but there are limited data on the stability of micronutrients in whole blood. OBJECTIVES: The objective was to investigate the effects of delayed processing of whole blood on the stability of 25 micronutrient and selected clinical biomarkers. METHODS: Blood from 16 healthy adults was collected into EDTA, lithium heparin (LH), or serum tubes. Samples were processed within 2 hours of collection ("2-hour processed") or mailed overnight (boxed with frozen cold packs) before processing ("24-hour processed"). Micronutrient and clinical biomarker concentrations were quantified with validated methods. The concentration percentage difference between the 2- and 24-hour processed samples was calculated and was compared against quality specifications determined from intra- and interindividual variations. RESULTS: All analytes had a sample type where the percentage difference concentration between 2-hour and 24-hour processed samples was ≤4% and was acceptable based on calculated limits, including for biomarkers of vitamin A, vitamin D, thiamin, folate, vitamin B-12, iron (ferritin), and zinc status and for selected clinical markers, C-reactive protein, HDL and total cholesterol, and triglycerides. EDTA plasma vitamin C was lower compared to the 2-hour processed sample (geometric mean, 43%; 95% CI: 36%-49%). Pyridoxal-5-phosphate (vitamin B-6 biomarker) decreased, with differences from the 2-hour processed samples of -8% (95% CI: -13% to -2%) and -14% (95% CI: -18% to -9%) in LH plasma and serum, respectively. CONCLUSIONS: In blood collected from adult participants, delayed processing of chilled whole blood for 24 hours did not materially affect the measured concentrations of the majority of micronutrients and selected clinical biomarkers. This suggests that for these analytes, adherence to a 2-hour processing protocol may be unnecessary. This knowledge is valuable and may help to simplify logistics for sample transport and processing of blood samples for micronutrient status assessment.

Low-dose thiamine supplementation of lactating Cambodian mothers improves human milk thiamine concentrations: a randomized controlled trial.

BACKGROUND: Infantile beriberi-related mortality is still common in South and Southeast Asia. Interventions to increase maternal thiamine intakes, and thus human milk thiamine, are warranted; however, the required dose remains unknown. OBJECTIVES: We sought to estimate the dose at which additional maternal intake of oral thiamine no longer meaningfully increased milk thiamine concentrations in infants at 24 wk postpartum, and to investigate the impact of 4 thiamine supplementation doses on milk and blood thiamine status biomarkers. METHODS: In this double-blind, 4-parallel arm randomized controlled dose-response trial, healthy mothers were recruited in Kampong Thom, Cambodia. At 2 wk postpartum, women were randomly assigned to consume 1 capsule, containing 0, 1.2 (estimated average requirement), 2.4, or 10 mg of thiamine daily from 2 through 24 weeks postpartum. Human milk total thiamine concentrations were measured using HPLC. An Emax curve was plotted, which was estimated using a nonlinear least squares model in an intention-to-treat analysis. Linear mixed-effects models were used to test for differences between treatment groups. Maternal and infant blood thiamine biomarkers were also assessed. RESULTS: In total, each of 335 women was randomly assigned to1 of the following thiamine-dose groups: placebo (n = 83), 1.2 mg (n = 86), 2.4 mg (n = 81), and 10 mg (n = 85). The estimated dose required to reach 90% of the maximum average total thiamine concentration in human milk (191 µg/L) is 2.35 (95% CI: 0.58, 7.01) mg/d. The mean ± SD milk thiamine concentrations were significantly higher in all intervention groups (183 ± 91, 190 ± 105, and 206 ± 89 µg/L for 1.2, 2.4, and 10 mg, respectively) compared with the placebo group (153 ± 85 µg/L; P < 0.0001) and did not significantly differ from each other. CONCLUSIONS: A supplemental thiamine dose of 2.35 mg/d was required to achieve a milk total thiamine concentration of 191 µg/L. However, 1.2 mg/d for 22 wk was sufficient to increase milk thiamine concentrations to similar levels achieved by higher supplementation doses (2.4 and 10 mg/d), and comparable to those of healthy mothers in regions without beriberi. This trial was registered at clinicaltrials.gov as NCT03616288.

Erythrocyte transketolase activity coefficient (ETKAC) assay protocol for the assessment of thiamine status.

Vitamin B1 (thiamine) is an essential nutrient that acts as a cofactor for a number of metabolic processes, particularly in energy metabolism. Symptoms of classic thiamine deficiency are recognized as beriberi, although clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult. Therefore, reliable biomarkers of thiamine status are required. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). The ETK activity assay as an indirect, functional marker of thiamine status has been used for over 50 years. The ETK activity assay provides a sensitive and specific biomarker of thiamine status; however, there is a lack of consensus over the cutoffs for deficiency, partly due to a lack of assay harmonization. Here, we provide a step-by-step protocol for the measurement of ETK activity and the calculation of the ETK activity coefficient, including detailed explanations of equipment and chemicals required and guidance for quality control procedures. Harmonization of the protocol will provide the basis for the development of internationally recognized cutoffs for thiamine insufficiency. The establishment of quality control materials and a quality assurance scheme are recommended to provide reliability. This will ensure that the ETK activity assay remains an important method for the assessment of thiamine status.