Thiamine has a crucial role in energy production, and consequently thiamine deficiency (TD) has been associated with cardiac failure, neurological disorders, oxidative stress (lactic acidosis and sepsis) and refeeding syndrome (RFS). This review aims to explore analytical methodologies of thiamine compound quantification and highlight similarities, variances and limitations of current techniques and how they may be relevant to patients.
An electronic search of Medline, PubMed and Embase databases for original articles published in peer-reviewed journals was conducted. MethodsNow was used to search for published analytical methods of thiamine compounds. Keywords for all databases included “thiamine and its phosphate esters”, “thiamine methodology” and terms related to critical illness. Enquiries were also made to six external quality assurance (EQA) programme organisations for the inclusion of thiamine measurement.
A total of 777 published articles were identified; 122 were included in this review. The most common published method is HPLC with florescence detection. Two of the six EQA organisations include a thiamine measurement programme, both measuring only whole-blood thiamine pyrophosphate (TPP). No standard measurement procedure for thiamine compound quantification was identified.
Overall, there is an absence of standardisation in measurement methodologies for thiamine in clinical care. Consequently, multiple variations in method practises are prohibiting the comparison of study results as they are not traceable to any higher order reference. Traceability of certified reference materials and reference measurement procedures is needed to provide an anchor to create the link between studies and help bring consensus on the clinical importance of thiamine.
The role of thiamine and more specifically thiamine pyrophosphate (TPP), the active form of what is commonly known as vitamin B1, in carbohydrate metabolism is well documented. However, a broader understanding of the biological role(s) of thiamine outside of carbohydrate metabolism is not yet available. Thiamine is a water-soluble vitamin which is passively absorbed in the small intestine at high concentrations or actively absorbed through thiamine transporter proteins at lower concentrations . It exists in multiple forms through the addition of one or more phosphate groups. These include the non-phosphorylated or free thiamine, thiamine monophosphate (TMP), TPP or diphosphate (TPP) and thiamine triphosphate (TTP) (Figure 1). Collectively, these will be referred to here as thiamine compounds.
The human adult can store around 30 mg of thiamine in muscle tissue, liver and kidneys . These stores can however become depleted in as little as 18 days after the cessation of thiamine intake , . This leads to a variety of clinical features that are often vague and sometimes non-specific , , . In recent years, there has been increasing recognition of its importance in neurological function as well as the discovery of new thiamine adenine nucleotide species , , , , . A greater understanding of human thiamine biochemistry is highly desirable and clinically relevant as thiamine deficiency (TD) is still reported worldwide. Despite long-term thiamine fortification of a variety of common household foods (meats, vegetables, milk products, cereals and whole grains), TD continues to be present even in modern, western societies , , , .
Classically, TD causes the disease known as beriberi, but this disease is really one of several thiamine-deficiency-related conditions, which may occur concurrently. Key contributors to TD include hospitalisation, moderate to severe alcohol consumption, and improper nutritional intake, e.g. a high carbohydrate load with restricted vitamin and mineral ingestion. Age, as well as co-morbidities, such as cardiac and liver dysfunction, surgery (e.g. bariatric), lactic acidosis, sepsis, trauma and refeeding syndrome (RFS) may also be associated risk factors for TD , , , , . All of these issues are commonplace in critically ill patients admitted to an intensive care unit (ICU).
Patients presenting to an ICU are often immobile and in an unconscious state. These populations frequently require nutritional support typically prescribed through parenteral or enteral administration. Despite this, the bio-availability of thiamine may be reduced due to fluid loss, increased metabolic demand, secondary to intensive care treatments or the development of RFS upon commencing parenteral or enteral nutrition. RFS itself is a multifaceted condition with numerous pathophysiological features and a similar cohort presentation to TD. The associated biochemistry of RFS is complex with the common biochemical profiles of low serum concentrations of magnesium, phosphate, potassium and thiamine all considered indicators of RFS , . Identifying RFS and TD therefore requires keen observation and a sound knowledge of potential risk factors, complemented with appropriate biochemical markers.
The significance of thiamine measurements in those at risk of RFS in the ICU may be more important than previously thought as states of subclinical deficiency or relative deficiency may also exist. If this is true, then additional research questions might include:
Should thiamine be routinely measured in hospitalised patients to assess their status?
If this is so, should the measurand be total thiamine (where all thiamine compounds are reduced to form free un-phosphorylated thiamine), or a profile of all thiamine compounds to assess the biological availability of each compound?
Finally, what current methodologies allow for the accurate measurement, and therefore clinical interpretation, of thiamine?
The aim of this review is to explore analytical methodologies of thiamine compound quantification and highlight similarities, variances and limitations of current techniques. A brief history of the vitamin and an examination of the relationship between levels of thiamine compounds in the blood of critically ill patients will also be presented.
The study objectives were achieved by electronically searching the Medline, PubMed and Embase databases for original articles published in peer-reviewed journals with no limitations on year of publication. The MethodsNow website was also used as a tool to search for published methods of thiamine compound quantification in a variety of matrices . Search keywords for all databases included “thiamine and its phosphate esters”, “thiamine methodology” and terms related to critical illness (Table 1). Manual searching of articles, including scanning the reference lists of cited publications, was also performed to avoid omissions. Overall, 777 papers were identified (723 using these databases, 54 through manual searching). A title scan of these papers reduced the total to 234 which was further reduced to 160 after the examination of abstracts and the removal of duplicate results. Of these 160 papers, 122 were included in this review.
|Embase PubMed Medline||– Critically ill OR Critical illness OR Critically ill patients OR Critical Care OR Intensive care OR Intensive care unit
– Thiamine OR Thiamin OR Vitamin B1 OR Anurine OR Thiamine monophosphate OR Thiamine pyrophosphate OR Thiamine diphosphate OR Thiamine triphosphate OR Thiamine deficiency OR Thiamine ester OR Thiamine derivative
– Peer reviewed
|MethodsNow||– Thiamine OR Thiamin OR Vitamin B1 OR Anurine OR Thiamine monophosphate OR Thiamine pyrophosphate OR Thiamine diphosphate OR Thiamine triphosphate OR Thiamine deficiency OR Thiamine ester OR Thiamine derivative
|– HPLC OR Florescence OR UV OR Ultra violet, OR mass spectrometry
– Urine OR Serum OR Plasma OR Blood OR Whole Blood OR Erythrocytes OR Washed erythrocytes
To further examine the current status of the analytical methods, enquiries were made to six EQA organisations to ascertain the inclusion of a thiamine related compound in their programme. Of these, two were found to include thiamine in the form of whole-blood TPP: The Dutch Foundation for Quality Assessment in Medical Laboratories (SKML) and The Royal College of Pathologists Australasia Quality Assurance Programs (RCPAQAP). These EQA programmes co-operate by using a common lyophilised material, purchased in bulk and distributed to participating laboratories in numerous countries.
The RCPAQAP and SKML schemes consist of a sample set of six linearly related levels analysed twice per cycle with two cycles each year , . All participants are presented with a survey to classify each component of their method. This survey information is divided into four categories: analytical principle, measurement system, reagent source and calibrator source. After each sample set, participants are provided with a survey report, indicating their performance in each of the method classifications.
Both agencies provided de-identified result sets and median values for their latest thiamine programme cycles for the purpose of this review. Results from the RCPAQAP Cycle 33 and SKML Cycles 20151 and 20152 programmes were used to assess the performance of current thiamine methodologies. As the results provided represent an identical batch of lyophilised sample material, the individual reported results were combined to generate an overall performance of the participating laboratories. For note, prior to analysing these results each individual cycle was subject to a one-way ANOVA test to ensure no population differences occurred among the cycles (p>0.05).
Early work on thiamine
The discovery of thiamine and indeed vitamins in general began with Eijkman’s early studies of the causes of beriberi in Java . Eijkman originally sought a bacterial cause of the disease, a logical conclusion at the time, as diseases arising from nutrient deficiency had not yet been recognised . The key discovery in Eijkman’s research occurred when the similarities between the symptoms of polyneuritis in his laboratory chickens and the polyneuritis occurring in beriberi were noticed. The chicken polyneuritis was traced to an accidental change in diet (of polished rather than unpolished rice) and when this was reversed, the birds recovered in a few days. Eijkman incorrectly concluded that the polished rice contained a toxic starch which was the cause of the leg weakness whilst the bran and germ of the whole rice neutralised this toxin , . Nowadays, it is understood that the polished white rice feed was deficient in thiamine, whilst the bran and germ in the unmilled brown rice was thiamine rich. Although Eijkman’s original hypothesis was incorrect, he was jointly awarded the Nobel Prize in Physiology and Medicine in 1929 with Sir Frederick Hopkins for the discovery of essential nutrient factors (vitamins) needed in animal diets to maintain health .
It was Gerrit Grijns (Eijkman’s successor) who correctly identified the disease as a lack of vital nutrients (and not the work of a toxin) in 1901 and attempted to isolate the missing nutritional element by introducing minerals and fats to the diets of the diseased chickens. Neither proved effective . Grijns’ later success in curing the condition using mung beans led to his conclusion that the underlying cause was a deficiency in a compound or compounds, as yet unidentified, present in some foods and absent from others . Despite Grijns’ pioneering research, his contributions were largely overlooked. His work, however, allowed for the emerging scientific movement of nutritional deficiencies to progress. Notable attributions that stemmed from both Eijkman and Grijns’ works include the discovery of other B vitamins and the coining of the term ‘vitamin’ (from vital amine) by Funk in 1912 (although many vitamins were later shown to not be amines) and the eventual isolation of crystallised thiamine in 1926 by Jansen and Donath , .
Physiology of thiamine
In the 1920s–1930s, thiamine was still known as either anti-neuritic vitamin or vitamin B1 as it was the first of the B complex vitamins to be discovered. Jansen, as well as many others, was not content with that nomenclature and in 1935 proposed the name aneurin (from anti-polyneuritisvitamin) , . As Jansen was half of the team that first isolated the crystalline structure, the rights to name the vitamin seemed appropriate. However, a year later, Williams succeeded in identifying the chemical structure and synthesising it , . In 1938, Williams formally introduced the term thiamin due to the fact it was a sulphur-containing vitamin . Whilst some older tests use the term anurine, today it is more commonly presented as thiamine (including the addition of an ‘e’ from the original name).
The work of Eijkman, Hopkins, Grijns and others brought vitamins to the forefront of health research and with it evidence of the existence of nutrient deficiency as a cause for disease. On a broader scale, this (much later) resulted in the addition of vitamins in foodstuffs, the existence of vitamin supplements as preventative measures to beriberi (vitamin B1 deficiency), rickets (vitamin D deficiency), scurvy (vitamin C deficiency) and pellagra (vitamin B3 deficiency), as well as vitamin recommended dietary intakes (RDI) , , , , , .
The distribution of thiamine compounds in the body varies considerably. After ingestion, free thiamine is converted to TPP by thiamine pyrophosphokinase and is then utilised by numerous metabolic pathways as a cofactor. TPP can also be further phosphorylated or dephosphorylated to form any of the thiamine compounds (Figure 1). Gangolf et al.  and Tallaksen et al.  conducted extensive studies on human cells and body fluids which determined that the most abundant intracellular thiamine species was TPP. TMP and free thiamine were the only detectable species in the extracellular fluid. The majority of TPP in the body is found in erythrocytes and accounts for approximately 80% of the total bodies stores , , , . There is no consensus on reference intervals for thiamine compounds in the human body. As an example published reference intervals for whole-blood TPP range from a lower limit of 63–105 nmol/L to an upper limit of 171–229 nmol/L , , , , , . Reference intervals for the less abundant compounds in whole blood and serum have also been established each with similar variations in their lower and upper limits , , , , , , .
Clinical practise publications related to nutritional supplementation state that patients with suspected or at risk of developing TD should be provided with thiamine , , . Malnutrition (starvation or reduced intake, increased loss or impaired absorption), alcoholism, bariatric surgery, refeeding, congestive heart failure, lactic acidosis, neuropathy, renal failure with dialysis and the critically ill are all risk factors for the development of TD. However, there is no consensus on the dose, frequency or duration of supplementation. For mild symptoms, 20 mg a day up to 100 mg 2 or 3 times a day has been suggested. More advanced symptoms include intravenous doses of 50 mg a day to >500 mg twice a day. These recommendations are dependent on the risk factor, with alcoholism and bariatric surgery calling for higher frequency and larger doses, as there is the potential of impaired absorption, compared to cardiac and renal failure.
Thiamine’s function as a cofactor, in the form of its diphosphate ester TPP, is the most broadly recognised physiological role. TPP is a cofactor for numerous enzymatic reactions including pyruvate dehydrogenase (PDH), transketolase, α-ketogluterate dehydrogenase (KGDH) and branched-chain α-keto acid dehydrogenase (BCKDH) , , , . Its involvement in these metabolic pathways and its intracellular abundance has made TPP a popular analyte for assessing thiamine status. However, there are also proposed non-cofactor roles of thiamine compounds within the immune system, gene regulation, oxidative stress response, cholinergic activity, chloride channels and neurotransmission , , , , , , .
The physiology of thiamine compounds regarding synthesis, transport and energy metabolism has been covered elsewhere and will not be reviewed here. For the interested reader, papers by Betterndorf , Lonsdale  and Manzetti  provide excellent overviews of thiamine in both cofactor and non-cofactor roles. Recent discoveries have also identified thiamine containing adenine nucleotides, but as there is little known in regard to their function and mechanisms they will not be discussed further in this review .
Pathophysiology related to TD
The diphosphate esters of thiamine are involved in numerous enzymatic reactions. In the mitochondria PDH, KGDH and BCKDH are heavily involved in the production of energy in the tricarboxylic acid cycle (TCA), or the Krebs cycle, feeding reduced hydrogen carrier nicotinamide adenine dinucleotide (NAD) into the electron transport chain for adenosine triphosphate (ATP) production (Figure 2). Transketolase is also involved in energy production oxidising two reactions in the pentose phosphate pathway transporting carbohydrates for glycolysis. A deficiency in thiamine can potentially lead to a decline in the activity of these TPP-dependent enzymes, causing a decreased energy yield or forcing alternate reactions to ensue.
Beriberi is the clinical disease which manifests from TD and presents in three forms. Genetic beriberi is a rare condition that prevents the body from absorbing thiamine. The two more commonly acquired forms of the disease are wet and dry beriberi. Wet beriberi affects the cardiovascular system and can cause cardiac failure. Dry beriberi damages the peripheral nervous system and can lead to muscle paralysis. Dry beriberi may co-present with neuropathies such as Wernicke’s encephalopathy and Korsakoff’s syndrome . Wernicke-Korsakoff syndrome may occur if both neurologic and psychiatric symptoms are present.
The clinical symptoms related to beriberi are associated with a reduction in the mitochondrial energy production leading to cardiac and cerebral susceptibility. Peripheral vasodilation, biventricular myocardial failure, volume overload, tachycardia, depression of left ventricular function with low ejection fraction, sudden onset cardiac failure and acute renal failure are just some of the signs of wet beriberi , , . In contrast, signs of dry beriberi include ophthalmoplegia, ataxia, confusion, memory loss and confabulation in chronic settings , , . These symptoms seem to be variable depending on age and the spectrum of the deficiency and may only precipitate during periods of increased energy demand or metabolism in individuals with only a subclinical deficiency. Although cases of beriberi are somewhat of a rarity in modern medicine, there are still populations that are at risk of developing TD. These include patients post-gastric surgery, the elderly, alcoholics, malnourished, diabetics, prolonged hospitalisation, severe fluid loss or fluid replacement therapy , , , , , , .
Considerations of thiamine status in critical illness is not a new approach. Studies of low thiamine compound levels include association with oxidative stress, sepsis, metabolic acidosis, cardiac failure, and neurodegeneration , , , , . The prevalence of TD in the ICU, hospitals and the elderly (>76 years of age) has also been examined , , . A retrospective study by Cruickshank et al.  observed a 20% prevalence of TD in intensive care patients requiring nutritional support. A mortality rate of 72% was seen in patients with TD compared to 50% in the non-TD group from 158 patients.
In the intensive care and general hospital setting, TD has been linked to specific presentations including metabolic acidosis, sepsis and septic shock, cardiac failure and surgery, neurodegeneration, and RFS; each of these will be discussed in turn below.
No study has been able correlate low thiamine levels with oxidative stress or lactic acidosis in the general ICU population. However, a study by Donnino et al.  reported a significant negative correlation between lactic acidosis and thiamine levels within a sub population without liver dysfunction. Moskowitz et al.  also showed a negative correlation on lactate and thiamine levels in patients with diabetic ketoacidosis. Furthermore, there have been several reported cases where a metabolic acidosis has been corrected with thiamine supplementation , , , .
Sepsis and septic shock
There is little evidence to support or reject thiamine supplementation in the treatment and prevention of sepsis and septic shock. Several studies have been described but the therapeutic role of thiamine is still inconclusive.
A retrospective study by Marik et al.  on the use of vitamin C with thiamine and hydrocortisone for the treatment of sepsis and septic shock showed a significant difference in mortality between the treated (n=47) and controlled groups (n=47). This suggests that the intervention may reduce mortality of patients with severe sepsis and septic shock.
Donnino et al.  observed no differences in lactate levels, severity of illness or mortality between the thiamine treatment (n=43) and control groups (n=45). However, a sub population for patients with baseline TD was also examined and a significant difference in lactate levels at 24 h and time to death was observed in this group.
A post-hoc analysis was performed by Moskowitz et al.  on this study to identify whether thiamine supplementation reduced the severity of kidney injury in septic shock. A significant difference was seen for the requirement of renal replacement therapy between the control and intervention groups (p=0.04) indicating that thiamine supplementation may reduce the risk of sepsis associated kidney injury. However, no difference was observed between the groups for in-hospital mortality (p=0.45).
The prevalence of TD in sepsis and septic shock has also been determined. A study on patients with sepsis in the ICU reported a TD prevalence of 10% upon admission which increased to 20% within the first 72 h post-admission . This study improved on a retrospective study by aforementioned Cruickshank paper by incorporating direct measurement techniques using high-pressure liquid chromatography (HPLC). A more recent study by Costa et al.  on septic shock also concluded that the prevalence of TD in the ICU was 20% upon admission but increased to 71.3% during the course of the study.
Cardiac failure and surgery
There is an association but no clear relationship between TD and cardiac failure. Ahmed et al.  reviewed the prevalence of TD in cardiac failure patients reporting an overall prevalence of 33%–91% in those that are hospitalised and 21%–27% in a consortium of inpatients and outpatients with cardiac failure. Multiple studies have shown improvement with supplementation whilst others show no difference between the disease and non-disease groups , , , , , .
A perspective observational study by Donnino et al.  showed a statistically significant difference in thiamine status before and 24 h post-cardiac surgery. The enrolled participants were specific to a relatively healthy population prior to surgery, as they were non-emergency operations with a rapid recovery prognosis. For this reason the study could not concluded that supplementation is warranted in critically ill and post-surgery populations.
However, they did highlight that physical stress, even in healthy populations, can cause a significant change in the thiamine status of a patient.
A perspective, double blind, randomised, placebo controlled, single centre trial by Berger et al.  investigated the outcomes of early antioxidant supplementation in the critically ill. The supplementation regime encompassed several micronutrients (100 mg of thiamine) for 5 days. A cardiac surgery cohort was also included. The study concluded that the intervention did not reduce early organ dysfunction but there was a significant reduction in the inflammatory response in cardiac and trauma patients.
Two double-blind, randomised, placebo-controlled studies (200- and 300-mg doses) were performed with a main endpoint of post-cardiac surgery blood lactate levels , . Both studies concluded no significant differences in blood lactate levels or clinical outcome between intervention and placebo groups. However, significantly higher blood thiamine levels were seen in the intervention group in both studies.
The neurodegeneration patterns of TD in Wernicke’s encephalopathy and Korsakoff’s syndrome (and the joint symptoms of Wenicke-Korsakoff’s syndrome) share some similarities with other neurodegenerative diseases such as Alzheimer’s disease , . All three present with reduced brain TPP, KGDH, PDH and transketolase concentrations, reduced glucose metabolism as well as the presence of twisted tau protein fibres, commonly referred to as tangles , , , . Studies have hypothesised that a decrease in brain thiamine compounds can lead to a cascade of reactions ultimately leading to neuronal cell death through excitotoxicity, inflammation and oxidative damage , , , . Recent comprehensive publications have reviewed the role of TD in impaired oxidative metabolism, excitotoxicity and blood-brain barrier alterations causing neurodegeneration , , .
Interestingly, only selective areas of neurodegeneration in the brain due to TD have been reported , , , , . This has led to theories that the neurodegeneration from TD is due to the localisation of either thiamine-dependent enzymes, with particular attention to KDGH, or alterations in the phosphokinase or phosphotransferase enzymes required to maintain thiamine brain homeostasis . One study has shown that thiamine supplementation does not significantly increase brain thiamine compound levels . Yet there have been reports that thiamine administration reduces and even reverses motor-sensory polyneuropathy in Wenicke-Korsakoff’s syndrome and post-gastrectomy , . This highlights that brain thiamine homeostasis by thiamine specific transporters, enzymatic phosphorylation and de-phosphorylation is still not fully understood. Verification and identification of specific enzymes and mechanisms, such as thiamine monophosphatase and thiamine triphosphosynthase, is yet to be achieved .
Besides studies into the levels of thiamine compounds in Wernicke’s encephalopathy, Korsakoff’s syndrome and in the brains of Alzheimer’s patients, there is little research in the area of neurodegeneration associated with TD in the critically ill. The neurological complications that arise from TD may be subclinical in the critically ill due to the latent nature of the deficiency. These symptoms may only become apparent once periods of increased energy requirement or decreased nutritional intake are encountered. Consequently these may not be routinely checked for upon admission and may be detrimental to the patient’s outcome.
Refeeding syndrome (RFS)
RFS is thought to be caused by the sudden shifts of molecules from the extracellular to intracellular space leading to serum nutrient displacement. During periods of starvation the body decreases the release of insulin due to the cessation of carbohydrate ingestion . Fats and proteins become the sole energy source where reduced intracellular phosphate levels ensue , . Upon recommencing carbohydrate nutrition insulin is secreted, stimulating cellular uptake of serum electrolytes as well as the reactivation of numerous pathways, including glycolysis, promoting uptake of serum thiamine and magnesium. Severe hypophosphataemia, hypokalaemia, hypomagnesaemia and TD are the predominant pathophysiological outcomes of these fluid shifts. However, these biochemical abnormalities are also present in sepsis, post-gastric surgery, post-trauma as well as liver disease to name but a few presenting conditions.
Clinical features of RFS are also non-specific but can include respiratory failure, cardiac failure, arrhythmias, seizures, coma or sudden death . A systematic review of 27 RFS case studies highlighted the inconsistencies of reported signs and symptoms . These reports emphasise the clinical spectrum of this disorder indicating that not all cases will present with RFS from classical malnutrition as some might be asymptomatic. Other clinical features that can be associated but not limited to RFS, which are also common place in the ICU, include fluid retention and hypotension as well as heart, brain and liver injury. Overall, its prevalence in the ICU is estimated at 34% .
As the human body does not store an excessive amount of thiamine, and RFS is known to be caused by a state of nutritional starvation, patients in intensive care are already likely to have some degree of TD. As glycolysis is re-engaged when initiating refeeding the requirement of thiamine-dependent enzymes intensifies, exhausting any thiamine stores available, potentially leading to the associated metabolic and neurological complications of TD. Although there is no direct evidence that links thiamine supplementation to improving the status of RFS, recommendations from case reports, reviews and guidelines all support thiamine administration prior to commencing and during the course of refeeding , , , , , , . In addition, parenteral or enteral refeeding is one of the main risk factors associated with RFS, along with alcoholism, post-bariatric surgery, cancer and anorexia . These clinical situations make the diagnosis of RFS challenging as individuals in intensive care do not always present with typical biochemical profiles.
Methods for the measurement of thiamine compounds
The therapeutic benefit of thiamine supplementation is still being debated with no consensus on patient groups, dosage or the duration of supplementation. This divergence is fundamentally due to discordant results from inconsistencies in methodologies. Coordinating an agreement on the role of thiamine compounds in the critically ill can only be attained with evidence resulting from methods deemed fit for the purpose for thiamine quantification. This is achievable by the standardisation of thiamine quantification practises including the implementation of a certified reference material and reference method to which all current methods can be made traceable to.
The analytical techniques for thiamine compounds are discussed in this section in relation to the total testing process. This encompasses the pre-analytical, analytical and post-analytical phases based on specific considerations in order to provide reliable thiamine results to the clinician. Important considerations across the total testing process for direct measurement of thiamine compounds can be found in Table 2.
|Pre-analytical||– Sample stability can be an issue if not stored correctly prior to analysis as thiamine is light sensitive. Samples should be stored at a minimum of –70°C and must be analysed within 48 h of sample preparation , 
– The choice of sample type must be appropriate for the analyte being measured
– Lack of standardisation introduces various storage conditions and stability parameters
|Analytical||– No certified reference material or reference method available
– No reliability on calibration accuracy
– The lack of standardisation introduces various methodologies without traceability
– Very few internal standard options are available
– Thiamine is unstable in alkaline conditions, however the intensity of the fluorescence increases with alkalinity, plateauing at pH 9.0 
– Column performance and longevity can be reduced under highly alkaline conditions
– Derivitisation requires hazardous oxidising chemicals such as cyanogen bromide and mercuric chloride . Although there are alternate chemicals which are less dangerous to replace it, such as potassium ferrocyanide (or hexacyanoferrate), this consequently reduces the fluorescence intensity of the trichromes , 
– Post-column derivatisation methods prolong column life and performance but requires a second chromatographic pump to mix the effluent with the derivitising agent to carry it to the detector
– Pre-column derivitisation provides sharper peaks, better resolution but requires equipment for a gradient elution , . This usually comes standard with modern HPLC equipment
– Imprecision must fit within at least the minimum range of biological variation
– No data on the commutability of calibrators and standards
|Post-analytical||– No standardised common reference intervals including those for gender, ethnicity and age
– No decision limit to determine therapeutic levels
The pre-analytical phase consists of specimen handling such as sample collection and storage. Special considerations for thiamine analysis in this phase include the type of sample collected, the collection tubes and the stability of the sample.
The choice of matrix depends on the thiamine compound of interest. TMP, TTP and thiamine are mainly concentrated in serum, plasma and urine , , . Thus, these matrices should be used when quantifying one or several of those analytes. Whole blood or washed erythrocytes can be used to measure TPP as it is predominantly found in red blood cells. However, whole blood is considered a more suitable sample type as it permits the quantification of all thiamine compounds and washing erythrocytes in saline can reduce the levels of the thiamine compounds in these cells . A study by Ihara et al.  showed no difference in the values obtained from sodium and potassium ethylenediaminetetraacetic acid (EDTA) or sodium heparin anticoagulant tubes when measuring from whole blood, plasma or washed erythrocytes.
There is limited data on the stability of thiamine compounds in biological fluids or chemical standards. Multiple publications describe the immediate storage of samples at either −20 or −70°C for the lysis of erythrocytes prior to analysis , , , , . Standard thiamine compound solutions have also been reported to be stable from 1 to 5 months when stored in either a whole blood or plasma matrix or in a 0.1-M hydrochloric acid (HCl) solution. The storage conditions of these standards ranged from 4 to −70°C , , , , . Overall, there is little evidence to suggest that these storage conditions improve analyte stability.
The analytical phase consists of all laboratory diagnostic processing. Methodologies, internal standard use, calibration, biological variation, quality assurance, and the traceability of results should all be considered. Thiamine compounds are present in nmol/L, thus require highly sensitive methodologies to detect and quantify them reliably. Currently there are several analytical techniques for the measurement of thiamine status in the human body. The transketolase activation test, or transketolase activity assay, and the pyrophosphate effect test are both indirect methods which monitor the status of TPP. Liquid chromatographic techniques, with a range of separation and detection methods, are direct methods of quantification and are the most commonly described , , .
Indirect measurement procedures for TPP only estimate the activity of the enzyme and are not quantitative. Transketolase testing was first introduced in the mid-1950s as an in vitro method for evaluating TD . It estimates the functional activity of the transketolase enzyme in erythrocytes by measuring the rate that hexoses are produced from the TPP-dependent pentose phosphate pathway; with a decreased result indicating TD . For the pyrophosphate effect test, a baseline measurement of transketolase activity is first recorded, then a second reading is taken after the addition of exogenous TPP. The difference is transcribed as a percentage increase in transketolase activity; with an increase of around 23% or more indicating TD. However, the upper limit of normal has been greatly debated with published values ranging from 15.5% to 40% of increased activity , .
Further considerations are required when interpreting results from these tests as they are known to be affected by patients with diabetes or liver dysfunction as well as any other condition impeding the synthesis of transketolase , . Recent end-of-cycle reports from the RCPAQAP and SKML EQA vitamin programmes indicate that no participating laboratory is currently reporting TPP values using indirect methodology , .
The first HPLC method for the direct measurement of thiamine compounds was reported in the late 1970s . Unlike indirect methods, serum, plasma and urine can be used to measure other thiamine compounds beside TPP , . This permits for the total thiamine status to be calculated or the biological availability of all thiamine compounds to be assessed. The limit of quantification for all thiamine compounds has been reported to be <3.0 nmol/L with linearity surpassing physiologically possible biological concentrations , , , .
The direct measurement of thiamine compounds by HPLC can be coupled with either fluorescence, ultra violet (UV), photo-diode array (PDA) detection and more recently, mass spectrometry (MS) , , . Separation can be accomplished using gradient or isocratic techniques, with or without ion-pairing chemicals and on a range of columns including octadecyl carbon chain silica based (C18), polymer, or amino. Sample preparation procedures include cloud point extraction (using the cloud phenomena of surfactants), solid phase extraction and derivitisation (which can occur pre- or post-column) , , , . There are also an array of mobile phase solutions and chemicals for sample preparation with variations in pH levels from 2 to 13, even when identical chemicals are utilised. An extensive flow chart of the differences and similarities for a wide variety of these published methods can be seen in Figure 3.
The use of HPLC with pre- or post-column derivitisation coupled with florescence detection is the most popular method for the detection and quantification of thiamine compounds. The principle of this method is the oxidation of the thiamine compounds in alkaline conditions into highly florescent trichromes , , . In the EQA programmes offered by SKML and RCPAQAP 89% of the participants utilised pre- or post-column derivitisation with florescence detection. Of the remaining EQA participants, 2% stated the use of liquid chromatography tandem mass spectrometry (LC-MS/MS), 3% were ambiguous in their report and 6% did not state the methodology used , .
There are three recent published methods for the measurement of thiamine, TMP and TPP using LC-MS/MS , , . Tashirova et al.  measured thiamine in plasma using an acetonitrile-based mobile phase and a C18 column. Puts et al.  measured TPP in whole blood using a methanol-based mobile phase and a C18 column. Lindhurst et al.  measured TMP and TPP from a post-mitochondrial supernatant using an acetonitrile based mobile phase and a Hypercarb porous graphite carbon column. These methods offer low limits of quantification and fast acquisition times (from 1.3 to 2.8 min). As mentioned earlier, this methodology is already present in some clinical laboratories with two laboratories in the latest SKML cycle listing their instrumentation as LC-MS/MS for TPP analysis. Although these methods only measure one or two analytes in the thiamine family from a single matrix type, the introduction of tandem MS provides a much-needed platform for a thiamine quantification reference method to be built upon.
The remainder of the analytical section will focus on the quality of the direct measurement of thiamine compounds.
A reference point is required to ensure certainty that all aspects of the method, including commercially available kits, are traceable through a documented unbroken chain of calibrations from an established hierarchy . The Joint Committee for Traceability in Laboratory Medicine (JCTLM) is an established database of higher order reference materials, measurement procedures and services. Currently, there is no higher-order reference method, or materials, listed in their database for thiamine and its esters on their website . This is a major limitation with all current thiamine quantitative methods. Without certified reference materials there is no anchor for commercially available secondary calibrators to be traceable to. Calibration accuracy is a necessity for the comparisons of results from multiple sources.
A top-down approach for standardisation of all thiamine compound methods in several matrix types (whole blood, serum and urine) will allow for analytical quality goals to be established. This will support target setting for thiamine EQA programmes and assess bias. It can then provide empirical evidence towards the effectiveness of thiamine supplementation, the establishment of more accurate reference intervals or recommended levels for healthy populations as well as expected values in critical illness.
In all, a reference anchor will allow for traceability, leading to standardisation and harmonisation between laboratory methods, and will support consistent clinical interpretations and permit the bridging of results from multiple methods.
The biological variability (BV) of an analyte can be applied to set quality standards for a method, such as imprecision, bias and total error. Fraser described this fine tuning of goals from minimal, desirable and optimal . The BV for TPP has been assessed with an individual CV of 4.8% and a between individual CV of 12.0% . These equate to desirable analytical goals of imprecision of 2.4%, bias of 3.2% and total error of 7.2%. Given the current variability between methods and lack of traceability, bias (and also total error) comparison goals are difficult to interpret for whole-blood TPP.
Based on the BV data, the analytical imprecision goals for TPP are <1.2% (optimal), 2.4% (desirable) and 3.6% (minimum). To compare actual performance to these quality goals for imprecision, cycles of both EQA programmes were reviewed, these being the RCPAQAP Cycle 33 (2016) and the SKML programme Cycle 20151 and 20152 (2015). As a whole, the programmes achieved a median coefficient of variance (CV) of 7.5% (n=925 reported results) , . Interestingly, 69% of the participants indicated that they were using the same commercial brand reagent and calibrator kits. Overall, these commercial kits achieved a median CV of 6.7%. A summary of the best, median and worst CV’s for each programme cycle and for the commercial kit results only is displayed in Table 3. With a large percentage of the population using identical reagent and calibrator kits, the major variables appear to be associated with instrumentation and sample preparation techniques. However, these commercial kits do not include an internal standard for TPP, thus the performance of these kits can still be considered as a major factor in the variability in the results obtained.
|CV%||RCPAQAP Cycle 33, 2016||SKML Cycle 20151/2, 2015||Combined programmes|
|Whole programme||Commercial kits||Whole programme||Commercial kits||Whole programme||Commercial kits|
|Participants||20||16 (80%)||63||41 (65%)||83||57 (69%)|
Data presented have had outliers removed. These data have been reproduced with permission of the RCPAQAP chemical pathology programme and SKML.
The inclusion of an internal standard in HPLC and LC-MS/MS methods is broadly recognised to be good laboratory practise as it improves assay imprecision as corrective action is taken for variations during sample preparation and instrument response. A survey of participants enrolled in the RCPAQAP whole-blood programme revealed that only one of nine respondents included an internal standard . Furthermore, from our literature search, only four publications reported the use of an internal standard , , , .
Non-isotopic internal standards, acetylaneurine, pyrithiamine and theobromine, were used in the three HPLC-florescent published methods , , . Only one of the three LC-MS/MS publications cited the use of an isotopic labelled (d3) internal standard . This is also the most recent LC-MS/MS publication. The absence of internal standards in thiamine compound methodologies is an unfortunate circumstance that may be caused by the difficulty in sourcing appropriate, commercially accessible, chemicals. This lack of availability is highlighted by each of these publications employing a different internal standard compound.
It must be noted that the commercial kits discussed above have very recently (2016) been updated to include an internal standard for TPP and have switched from a post-column to a pre-column derivitisation step. Whether this is attributed to the company’s own decision from published information or from consumer demand to improve accuracy and precision is not known . What can be acknowledged is that this is a necessity to improve the performance of these quantitative methods.
A further potential limitation not discussed in the literature is the commutability of thiamine compound reference materials. This applies to any non-patient material i.e. calibration standards, quality controls and EQA materials. A material is commutable when it behaves with the same numerical relationship as is seen with patient samples. This commutability needs to be demonstrated experimentally. Our review of the literature and product information related to thiamine did not locate any data that certified the commutability of calibration standards or quality controls used for thiamine compound quantification.
The RCPAQAP and SKML vitamin material is based on the relevant matrix (e.g. whole blood) but the material has undergone a range of manipulations including multiple analyte additions, mixing procedures and lyophilisation, any of which may affect commutability of the analyte. As this EQA material has not been formally verified for commutability the organisers specify that the material cannot be assumed to be commutable.
The commutability of calibration and also EQA material is an important consideration not to be overlooked as we move forward with improving the harmonisation and eventual standardisation of thiamine compound methods.
The post-analytical phase consists of the generation of reports and interpretation. This involves specifically how we interpret our thiamine compound result. To turn the numerical value into a clinically meaningful result we need an appropriate decision point(s) such as a reference interval. A survey of RCPAQAP participants measuring vitamins B1, conducted in 2012, found a general consensus towards reported reference intervals for vitamin B1 . The survey concluded that the apparent harmonisation of reference intervals was based upon their adoption from the reagent kit inserts, as 66% of the responding participants utilised a common commercially available reagent. This is considered as the lowest level of harmonisation by the Milan consensus statement for analytical performance specifications .
In the current literature, there are limited data on reference intervals based on gender or ethnicity , , . However, there is evidence of age-related differences . The elderly (>76 years of age) are known to have a reduced total thiamine compound concentration that can be 21%–26% lower than that of the middle aged , . A longitudinal study of over 300 participants spanning 3 years by Wilkinson et al.  examined a younger, healthy population (mean age 41.5 years) of 100 volunteers and compared it to an elderly population (mean age 76 years) of 221 participants. The study concluded that there was a significant difference between the young and old populations (p<0.001; Student’s unpaired t-test) and suggested that lower TPP levels in the elderly were related more to age than a co-existing illness. This is an additional concern when determining whether the lower thiamine compound levels are due to age or the disease state.
It should be noted that the elderly participants in the study described above were recruited from a general practise, not a hospital setting. Furthermore, the blood TPP results from the elderly sub-group were 117–142 nmol/L which is well within all published reference intervals of 63–229 nmol/L , , . Although this study provides clear evidence that there is an age-related decrease in TPP, the elderly population did not appear to be clinically thiamine deficient. A likely explanation would be that in a community setting for the elderly the co-morbidities are functional illnesses and thus less severe than in a nursing, full-time care or hospital setting. This is supported by an observational study be Lee et al.  who found a 14% prevalence of TD in elderly patients admitted to hospital from a nursing facility. This highlights that the predisposition to reduced TPP levels in the elderly increases the risk of TD in critical care scenarios.
Although the lack of agreement between analytical methods continues, a higher level common reference interval or decision limit for interpretation cannot be introduced. Therefore, reference intervals and expected values of disorders associated with TD cannot be generated nor broadly adopted. Further to such work, a critical result limit for immediate clinical notification and action could be identified.
A better understanding of TD and RFS in the critically ill is warranted as these appear to be disorders with a wide spectrum and the likelihood of sub-clinical deficiencies. There is currently no clear evidence as to which ICU populations are at risk of developing further complications from TD and RFS or whether any are asymptomatic. Investigations into thiamine compound levels in ICU populations is essential to understand the role of thiamine in these situations. A lack of consensus on the benefits of thiamine supplementation to prevent RFS and TD in the critically ill stems from absence of empirical evidence. A strong link between thiamine compound levels and TD and RFS is possible and may be likely but remains to be established. This evidence is paramount to provide consensus on thiamine supplementation leading to ICU-relevant protocols ensuring that these potentially important deficiencies are addressed.
To successfully investigate and predict clinical outcomes associated with TD, several key foundations are necessary; the capacity to quantify total thiamine status in biological fluids, universally accepted reference intervals for healthy populations and expected values for disorders associated with TD. For these to occur, standardisation of thiamine compound methodologies is required to allow for traceable results. This is currently unachievable without an accredited reference method and material.
We thank Mrs Sabrina Koetsier from the RCPAQAP and Dr Cas Weykamp from the SKML for their constant support, for providing de-identified cycle reports and for reviewing parts of this manuscript prior to submission. We would also like to thank the Australian government research training programme scholarship for awarding an Australian Postgraduate Award to J.T.B.C.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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