Individuals with type 2 diabetes mellitus (T2DM) are at increased risk of coronary artery disease (CAD). The C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene is associated with elevated plasma levels of homocysteine. The association of the MTHFR gene and the level of homocysteine with development of CAD has been studied in various population groups, including patients with T2DM, but the results have been variable. In practice, plasma homocysteine may be ordered as part of a screen for people with CAD or stroke, or who are at high risk for CAD or stroke but no other known risk factors. Testing of C677T polymorphism with or without elevated homocysteine is not recommended and has no clinical utility.
by Prof. Bakri Saeed and Dr Nisreen Mohammed
Type 2 diabetes mellitus and coronary artery disease
Type 2 diabetes mellitus (T2DM) is a major health problem throughout the world. It is a polygenic and multifactorial disease that is a major risk factor for cardiovascular disease. Cardiovascular disease (CVD) comprises coronary artery disease (CAD), also referred to as coronary heart disease (CHD), or ischemic heart disease (IHD), and cerebrovascular disease.
CAD due to atherosclerosis is a cause of significant morbidity and mortality, and is the leading cause of death worldwide. There are several risk factors for CAD. The well-stablished risk factors for CAD include diabetes mellitus, hypertension, smoking and dyslipidemia. There is growing interest in emerging risk factors for improved understanding of the mechanisms that underline cardiovascular disorders and CAD.
T2DM increases the risk for CAD by 2–4-fold compared to people without diabetes. CVD accounts for about 70% of deaths in people with diabetes. Identification and management of risk factors for CAD is an important aspect of management of diabetes mellitus.
Hyperhomocysteinemia and MTHFR polymorphism
Homocysteine is a sulfur-containing amino acid formed from demethylation of methionine. Methionine is the precursor to S-adenosyl methionine (SAMe) and is one of the essential amino acids. SAMe is a major methyl donor and is involved in numerous biological reactions. Homocysteine is metabolized by either remethylation to methionine or transsulfuration to cystathionine. The former reaction is catalysed by the vitamin B12-dependent methionine synthase. The latter reaction is catalysed by the enzyme cystathionine beta-synthase, which requires vitamin B6.
The methyl donor in the remethylation of homocysteine to methionine is 5-methyltetrahydrofolate. The 5,10-methylene-tetrahydrofolate reductase (MTHFR) enzyme catalyses the reduction of 5,10-methylene-tetrahydrofolate to 5-methyltetrahydrofolate. The enzyme requires B2 (riboflavin) as a cofactor (Fig. 1).
Therefore, hyperhomocysteinemia can result from reduced activity of the enzymes involved in homocysteine metabolism or from deficiency of the vitamins which are needed as cofactors in homocysteine metabolic reactions: folate, vitamin B6 and vitamin B12.
Several mutations in the MTHFR gene have been identified and some of them affect the activity of the enzyme. The commonest MTHFR gene mutation is a cytosine-to-thymidine substitution at nucleotide 677 (C677T), which changes alanine into valine, resulting in a thermolabile enzyme with impaired enzymatic activity and leading to hyperhomocysteinemia.
There are two copies of each gene. Therefore, an individual can be homozygous for the mutated gene or can be heterozygous, having one copy of the C677T variant and one normal copy. The C677T homozygous variant enzyme is thermolabile and demonstrates 70% reduced enzyme activity in vitro. The heterozygous C677T MTHFR enzyme has 35% reduced activity in vitro.
Worldwide, the frequency of MTHFR gene mutations varies among racial and ethnic groups, in Africa MTHFR gene polymorphism is markedly low (below 10%) for the C677T allele. In the European and Asian population, estimates of 18.6% and 20.8% were reported [1].
Association with CAD
In recent years hyperhomocysteinemia has been implicated as a risk factor for CAD, independent of other known risk factors. The primary mechanism by which homocysteine promotes atherosclerosis is by impairing endothelial function, which initiates the chain of events resulting in atherosclerotic plaque formation.
Numerous studies looked into the possible association between MTHFR genotypes and plasma homocysteine levels and the incidence of different MTHFR genotypes and hyperhomocysteinemia in CAD patients [2–5]. The results of these studies have been controversial. Several studies have shown the link between the MTHFR C677T gene polymorphism and the risk for CAD but many other studies failed to show association between MTHFR genotypes and plasma homocysteine levels and their role in CAD.
Previous studies in T2DM patients were also controversial. MTHFR polymorphism and hyperhomocysteinemia were shown to be predictors of cardiovascular events among diabetic patients [6, 7], whereas other studies failed to show a role for MTHFR polymorphic variants and homocysteine in increasing susceptibility to cardiovascular disease [8, 9].
Our study
We recently screened 226 consecutive patients with T2DM, <60 years of age, diagnosed according to WHO criteria. Of these, 113 had CAD confirmed by angiography and electrocardiography (ECG) and 113 had no evidence of CAD [10]. PCR and restriction fragment length polymorphism (RFLP) using Hinf1 restriction enzyme were used to determine MTHFR genotypes.
In our study, the T allele had a significant effect on homocysteine level (P value <0.05) and showed strong association with CAD among T2DM patients (odds ratio 6.2, P <0.0001).
Our study indicates that the C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinemia, and the two are independently associated with the presence of CAD in patients with T2DM.
Reasons for controversy
The outcome of these numerous studies and meta-analysis remained contradictory. There was no agreement on the association between MTHFR genotypes and plasma homocysteine levels or the incidence of different MTHFR genotypes and hyperhomocysteinemia in CAD patients.
Plasma homocysteine levels are dependent on interacting nutritional and genetic factors. Some studies suggested that people homozygous for MTHFR C667T polymorphism tend to have hyperhomocysteinemia in the context of low folic acid levels. Supplementation with the vitamins involved in homocysteine metabolism was found to lower plasma homocysteine levels.
Therefore, geographic heterogeneity, nutritional and environmental factors could affect the relationship between MTHFR genotypes and CVD risk in different populations.
Practical points
Homocysteine may be ordered as part of a screen for people with or at high risk of CAD or stroke, especially if there is family history of CAD or stroke but no other known risk factors, such as diabetes, smoking, hypertension, or dyslipidemia. Routine screening of homocysteine, like that of cholesterol, has not been recommended.
Plasma homocysteine concentration may be elevated in B12 and folate deficiency and its measurement has been suggested to give an early indicator of deficiency.
In new-born testing, greatly increased concentrations of homocysteine in the urine and blood suggests a diagnosis of homocystinuria and indicates the need for confirmation of the cause of raised levels.
Most laboratories report normal homocysteine levels in the blood between 5 and 15 µmol/L. Any measurement above 15 µmol/L is considered high.
However, it should be noted that normal levels will vary between ethnic groups and populations. Homocysteine levels increase with age, are lower in pregnancy and are influenced by drugs. These factors should be taken into consideration when interpreting results.
Testing of C677T polymorphism with or without elevated homocysteine is not recommended in patients with CAD or other diseases where MTHFR variants have been implicated, such as thrombophilia or recurrent pregnancy loss.
References
1. Schneider JA, Rees DC, Liu YT, Clegg JB. Worldwide distribution of a common methylenetetrahydrofolate reductase mutation. Am J Hum Genet 1998; 62: 1258–1260.
2. Chehadeh SWEH, Jelinek HF, Al Mahmeed WA, Tay GK, Odama UO, Elghazali GE, et al. Relationship between MTHFR C677T and A1298C gene polymorphisms and complications of type 2 diabetes mellitus in an Emirati population. Meta gene 2016; 9: 70–75.
3. Bickel C, Schnabel R, Zengin E, Lubos E, Rupprecht H, Lackner K, et al. Homocysteine concentration in coronary artery disease: Influence of three common single nucleotide polymorphisms. Nutr Metab Cardiovascular Dis 2017; 27(2): 168–175.
4. Yilmaz H, Isbir S, Agachan B, Ergen A, Farsak B, Isbir T. C677T mutation of methylenetetrahydrofolate reductase gene and serum homocysteine levels in Turkish patients with coronary artery disease. Cell Biochem Funct 2006; 24(1): 87–90.
5. Meisel C, Cascorbi I, Gerloff T, Stangl V, Laule M, Müller JM, et al. Identification of six methylenetetrahydrofolate reductase (MTHFR) genotypes resulting from common polymorphisms: impact on plasma homocysteine levels and development of coronary artery disease. Atherosclerosis 2001; 154(3): 651–658.
6. Lewis SJ, Ebrahim S, Smith GD. Meta-analysis of MTHFR 677C→T polymorphism and coronary heart disease: does totality of evidence support causal role for homocysteine and preventive potential of folate? BMJ 2005; 331(7524): 1053–1058.
7. Bennouar N, Allami A, Azeddoug H, Bendris A, Laraqui A, El Jaffali A, et al. Thermolabile methylenetetrahydrofolate reductase C677T polymorphism and homocysteine are risk factors for coronary artery disease in Moroccan population. J Biomed Biotechnol 2007(1); 80687.
8. Bahadır A, Eroz R, Türker Y. Does the MTHFR C677T gene polymorphism indicate cardiovascular disease risk in type 2 diabetes mellitus patients? Anatolian J Cardiol 2015; 15(7): 524–530.
9. Rahimi Z, Nomani H, Mozafari H, Vaisi-Raygani A, Madani H, Malek-Khosravi S, et al. Factor V G1691A, prothrombin G20210A and methylenetetrahydrofolate reductase polymorphism C677T are not associated with coronary artery disease and type 2 diabetes mellitus in western Iran. Blood Coagul Fibrinolysis 2009; 20(4): 252–256.
10. Mohammed NO, Ali IA, Elamin BK and Saeed BO. The association of methylenetetrahydrofolate reductase gene polymorphism and hyperhomocysteinaemia with coronary artery disease in Sudanese patients with type 2 diabetes. Poster at Focus 2017, Association of Clinical Biochemistry annual meeting.
The authors
Bakri Osman Saeed*1 PhD, MD, FRCPath, FRCP; Nisreen Osman Mohamed2 PhD
1Faculty of Medicine, Sudan International University, Khartoum, Sudan
2Ahfad Centre for Science and Technology, Ahfad University for Women, Khartoum, Sudan
*Corresponding author
E-mail: saeedbakri@hotmail.com
Prognostic value of molecular and imaging biomarkers in patients with supratentorial glioma
PURPOSE: We evaluated the relationship between 11C-methionine PET (11C-METH PET) findings and molecular biomarkers in patients with supratentorial glioma who underwent surgery.
METHODS: A consecutive series of 109 patients with pathologically proven glioma (64 men, 45 women; median age 43 years) referred to our Institution from March 2012 to January 2015 for tumour resection and who underwent preoperative 11C-METH PET were analysed. Semi-quantitative evaluation of the 11C-METH PET images included SUVmax, region of interest-to-normal brain SUV ratio (SUVratio) and metabolic tumour volume (MTV). Imaging findings were correlated with disease outcome in terms of progression-free survival (PFS), and compared with other clinical biological data, including IDH1 mutation status, 1p/19q codeletion and MGMT promoter methylation. The patients were monitored for a mean period of 16.7 months (median 13 months).
RESULTS: In all patients, the tumour was identified on 11C-METH PET. Significant differences in SUVmax, SUVratio and MTV were observed in relation to tumour grade (P<0.001). IDH1 mutation was found in 49 patients, 1p/19q codeletion in 58 patients and MGMT promoter methylation in 74 patients. SUVmax and SUVratio were significantly inversely correlated with the presence of IDH1 mutation (P<0.001). Using the 2016 WHO classification, SUVmax and SUVratio were significantly higher in patients with primary glioblastoma (IDH1-negative) than in those with other diffuse gliomas (P<0.001). Relapse or progression was documented in 48 patients (median PFS 8.7 months). Cox regression analysis showed that SUVmax and SUVratio, tumour grade, tumour type on 2016 WHO classification, IDH1 mutation status, 1p/19q codeletion and MGMT promoter methylation were significantly associated with PFS. None of these factors was found to be an independent prognostic factor in multivariate analysis.
CONCLUSION: 11C-METH PET parameters are significantly correlated with histological grade and IDH1 mutation status in patients with glioma. Grade, pathological classification, molecular biomarkers, SUVmax and SUVratio were prognostic factors for PFS in this cohort of patients. The trial was registered with ClinicalTrials.gov (registration: NCT02518061).
Expression of cell cycle regulators and biomarkers of proliferation and regrowth in human pituitary adenomas
PURPOSE: The pathogenesis of pituitary adenomas (PA) is complex. Ki-67, pituitary tumour transforming gene (PTTG), vascular endothelial growth factor (VEGF), cyclin D1, c-MYC and pituitary adenylate cyclase-activating peptide (PACAP) protein expression was analysed and correlated with tumour and patient characteristics.
METHODS: 74 pituitary tumour samples (48 non-functional PA, 26 functional PAs); immunohistochemical analysis of protein expression, retrospective analysis of MR images and in vitro analysis of octreotide treatment was carried out on GH3 cells.
RESULTS: PTTG expression was negatively associated with age and positively with PA size, regrowth and Ki-67 index. Cyclin D1 correlated with Ki-67 and tumour size. c-MYC negatively correlated with size of tumour and age, and correlated with PTTG expression. Somatostatin analogue treatment was associated with lower Ki-67, PTTG and cyclin D1 expression while T2 hypointense PAs were associated with lower PTTG, cyclin D1, c-MYC and Ki-67. In vitro analyses confirmed the effect of somatostatin analogue treatment on PTTG and cyclin D1 expression.
CONCLUSIONS: Interesting and novel observations on the differences in expression of tumour markers studied are reported. Correlation between Ki-67 expression, PTTG nuclear expression and recurrence/regrowth of PAs, emphasizes the role that Ki-67 and PTTG expression have as markers of increased proliferation. c-MYC and PTTG nuclear expression levels were correlated providing evidence that PTTG induces c-MYC expression in PAs and we propose that c-MYC might principally have a role in early pituitary tumorigenesis. Evidence is shown that the anti-proliferative effect of somatostatin analogue treatment in vivo occurs through regulation of the cell cycle.
Comparison of multiple tau PET measures as biomarkers in aging and Alzheimer’s Disease
The recent development of tau-specific positron emission tomography (PET) tracers enables in vivo quantification of regional tau pathology, one of the key lesions in Alzheimer’s disease (AD). Tau PET imaging may become a useful biomarker for clinical diagnosis and tracking of disease progression but there is no consensus yet on how tau PET signal is best quantified. The goal of the current study was to evaluate multiple whole-brain and region-specific approaches to detect clinically relevant tau PET signal. Two independent cohorts of cognitively normal adults and amyloid-positive (Aβ+) patients with mild cognitive impairment (MCI) or AD-dementia underwent [18F]AV-1451 PET. Methods for tau tracer quantification included: (i) in vivo Braak staging, (ii) regional uptake in Braak composite regions, (iii) several whole-brain measures of tracer uptake, (iv) regional uptake in AD-vulnerable voxels, and (v) uptake in a priori defined regions. Receiver operating curves characterized accuracy in distinguishing Aβ− controls from AD/MCI patients and yielded tau positivity cut-offs. Clinical relevance of tau PET measures was assessed by regressions against cognition and MR imaging measures. Key tracer uptake patterns were identified by a factor analysis and voxel-wise contrasts. Braak staging, global and region-specific tau measures yielded similar diagnostic accuracies, which differed between cohorts. While all tau measures were related to amyloid and global cognition, memory and hippocampal/entorhinal volume/thickness were associated with regional tracer retention in the medial temporal lobe. Key regions of tau accumulation included medial temporal and inferior/middle temporal regions, retrosplenial cortex, and banks of the superior temporal sulcus. Our data indicate that whole-brain tau PET measures might be adequate biomarkers to detect AD-related tau pathology. However, regional measures covering AD-vulnerable regions may increase sensitivity to early tau PET signal, atrophy and memory decline.
C-terminal fragments of the amyloid precursor protein in cerebrospinal fluid as potential biomarkers for Alzheimer disease
This study assesses whether C-terminal fragments (CTF) of the amyloid precursor protein (APP) are present in cerebrospinal fluid (CSF) and their potential as biomarkers for Alzheimer’s disease (AD). Immunoprecipitation and simultaneous assay by Western blotting using multiplex fluorescence imaging with specific antibodies against particular domains served to characterize CTFs of APP in human CSF. We demonstrate that APP-CTFs are detectable in human CSF, being the most abundant a 25-kDa fragment, probably resulting from proteolytic processing by η-secretase. The level of the 25-kDa APP-CTF was evaluated in three independent CSF sample sets of patients and controls. The CSF level of this 25-kDa CTF is higher in subjects with autosomal dominant AD linked to PSEN1 mutations, in demented Down syndrome individuals and in sporadic AD subjects compared to age-matched controls. Our data suggest that APP-CTF could be a potential diagnostic biomarker for AD.
Blood-based biomarkers for the identification of sports-related concussion
Sports-related concussions (SRCs) are common among athletes in the United States. Most athletes who sustain an SRC recover within 7 to 10 days; however, many athletes who sustain the injury do not recover as expected and experience prolonged, persistent symptoms. In this document, the authors provide an overview of the empirical evidence related to the use of blood-based brain biomarkers in the athlete population for diagnosis of SRCs, prognosis of recovery and return to play guidelines, and indications of neurodegeneration. The authors also provide a summary of research challenges, gaps in the literature, and future directions for research.
Brain biomarkers and pre-injury cognition are associated with long-term cognitive outcome in children with traumatic brain injury
BACKGROUND: Children with traumatic brain injury (TBI) are frequently at risk of long-term impairments of attention and executive functioning but these problems are difficult to predict. Although deficits have been reported to vary with injury severity, age at injury and sex, prognostication of outcome remains imperfect at a patient-specific level. The objective of this proof of principle study was to evaluate a variety of patient variables, along with six brain-specific and inflammatory serum protein biomarkers, as predictors of long-term cognitive outcome following pediatric TBI.
METHOD: Outcome was assessed in 23 patients via parent-rated questionnaires related to attention deficit hyperactivity disorder (ADHD) and executive functioning, using the Conners 3rd Edition Rating Scales (Conners-3) and Behaviour Rating Inventory of Executive Function (BRIEF) at a mean time since injury of 3.1 years. Partial least squares (PLS) analyses were performed to identify factors measured at the time of injury that were most closely associated with outcome on (1) the Conners-3 and (2) the Behavioural Regulation Index (BRI) and (3) Metacognition Index (MI) of the BRIEF.
RESULTS: Higher levels of neuron specific enolase (NSE) and lower levels of soluble neuron cell adhesion molecule (sNCAM) were associated with higher scores on the inattention, hyperactivity/impulsivity and executive functioning scales of the Conners-3, as well as working memory and initiate scales of the MI from the BRIEF. Higher levels of NSE only were associated with higher scores on the inhibit scale of the BRI.
CONCLUSIONS: NSE and sNCAM show promise as reliable, early predictors of long-term attention-related and executive functioning problems following pediatric TBI.
Biomarkers of stroke recovery: Consensus-based core recommendations from the Stroke Recovery and Rehabilitation Roundtable
The most difficult clinical questions in stroke rehabilitation are “What is this patient’s potential for recovery?” and “What is the best rehabilitation strategy for this person, given her/his clinical profile?” Without answers to these questions, clinicians struggle to make decisions regarding the content and focus of therapy, and researchers design studies that inadvertently mix participants who have a high likelihood of responding with those who do not. Developing and implementing biomarkers that distinguish patient subgroups will help address these issues and unravel the factors important to the recovery process. The goal of the present paper is to provide a consensus statement regarding the current state of the evidence for stroke recovery biomarkers. Biomarkers of motor, somatosensory, cognitive and language domains across the recovery timeline post-stroke are considered; with focus on brain structure and function, and exclusion of blood markers and genetics. We provide evidence for biomarkers that are considered ready to be included in clinical trials, as well as others that are promising but not ready and so represent a developmental priority. We conclude with an example that illustrates the utility of biomarkers in recovery and rehabilitation research, demonstrating how the inclusion of a biomarker may enhance future clinical trials. In this way, we propose a way forward for when and where we can include biomarkers to advance the efficacy of the practice of, and research into, rehabilitation and recovery after stroke.
Brain biomarkers of vulnerability and progression to psychosis
Identifying predictors and elucidating the fundamental mechanisms underlying onset of psychosis are critical for the development of targeted pre-emptive interventions. This article presents a selective review of findings on risk prediction algorithms and potential mechanisms of onset in youth at clinical high-risk for psychosis, focusing principally on recent findings of the North American Prodrome Longitudinal Study (NAPLS). Multivariate models incorporating risk factors from clinical, demographic, neurocognitive, and psychosocial assessments achieve high levels of predictive accuracy when applied to individuals who meet criteria for a prodromal risk syndrome. An individualized risk calculator is available to scale the risk for newly ascertained cases, which could aid in clinical decision making. At risk individuals who convert to psychosis show elevated levels of proinflammatory cytokines, as well as disrupted resting state thalamo-cortical functional connectivity at baseline, compared with those who do not. Further, converters show a steeper rate of grey matter reduction, most prominent in prefrontal cortex, that in turn is predicted by higher levels of inflammatory markers at baseline. Microglia, resident immune cells in the brain, have recently been discovered to influence synaptic plasticity in health and impair plasticity in disease. Processes that modulate microglial activation may represent convergent mechanisms that influence brain dysconnectivity and risk for onset of psychosis and thus may be targetable in developing and testing preventive interventions.
Steroid metabolism
All steroids share a cyclopentanoperhydrophenanthrene nucleus, with individual species varying according to the presence of different functional groups attached to this four-ring structure, as well as by the oxidation state of the rings. Cortisol structure is given as an example in Figure 1. In humans, the major sites of steroid hormone production are the adrenal gland and the gonads. Steroids are synthesized from cholesterol via a series of enzyme-catalysed steps (Fig. 2), which are under tight regulation in healthy individuals by feedback mechanisms involving the hypothalamus and anterior pituitary. Steroids have a wide range of physiological functions which are summarized in Table 1.
Adrenocortical carcinoma – a diagnostic challenge
There are many endocrine disorders that result in the improper synthesis of steroids, and one of the rarest and most severe is adrenocortical carcinoma (ACC). ACC is a malignancy of the adrenal cortex with an annual incidence of 1 or 2 cases per million [1]. The majority of ACC cases are sporadic and occur in the fifth or sixth decade of life and more commonly in women; although ACC can be associated with several familial syndromes including Li-Fraumeni, Beckwith-Wiedemann, Lynch syndrome and multiple endocrine neoplasia type 1 [2]. Functional steroid hormone-producing tumours occur in around two-thirds of cases [3], presenting with varied signs and symptoms of steroid overproduction, most commonly Cushing’s syndrome (cortisol excess) and hyperandrogenism. ACC can progress rapidly in some patients, therefore it is vital that it is distinguished from benign adrenal adenomas, as ACC has a 5-year survival rate of <50% [2]. A surgical cure is only possible if the carcinoma is detected in its localized stage, otherwise the median survival period is <15 months [4].
The diagnosis of ACC is challenging as there is no single diagnostic tool that is able to distinguish ACC from other adrenal masses, including benign adenomas with glucocorticoid or mineralocorticoid excess, phaeochromocytoma and non-functioning adenomas. Imaging alone is insufficient for diagnosis, as although patients with ACC almost always present with tumours ≥4 cm, the presence of a large mass only has a clinical specificity of 61% [5]. Additionally, whereas up to two-thirds of tumours are functional, less than half of ACC cases present with clinical signs of steroid overproduction [3], with a further proportion presenting with other symptoms including abdominal pain. However, a significant proportion are discovered incidentally [2].
The European Network for the Study of Adrenal Tumours (ENSAT) currently recommends that the initial biochemical work-up for suspected ACC includes measurement of serum cortisol (both basal and assessment of suppression after dexamethasone), dehydroepiandrostenedione sulphate (DHEAS), androstenedione, testosterone, 17-hydroxyprogesterone, estradiol and aldosterone (if the patient is hypokalemic or hypertensive). An alternative approach is to measure steroid metabolites in urine using gas chromatography-mass spectrometry (GC-MS); increases in the excretion of metabolites of the steroid precursors 11-deoxycortisol, 17-hydroxypregnenolone and pregnenolone have been shown to provide particularly high diagnostic utility in ACC. Unfortunately, urine steroid profiling is not commonly available in clinical laboratories owing to lengthy sample preparation and complex result interpretation. Further, serum 11-deoxycortisol, 17-hydroxypregnenolone or pregnenolone measurements are rarely performed either because of lack of demand, or specificity of the available immunoassays which may be subject to significant levels of cross-reactivity.
As a result of these limitations, the use of LC-MS/MS is increasingly being adopted to provide more specific steroid hormone measurements. An approach we have taken in our laboratory is to develop and fully evaluate a multiplexed LC-MS/MS method panelling 13 steroids in serum [6] to include many of the steroid synthetic pathway intermediates currently not available for ACC work-up.
Use of a serum steroid panel
The steroids included in our serum panel are highlighted in Figure 2 and are as follows:
Samples are prepared for analysis by an initial protein precipitation step to remove steroids from their binding proteins, followed by liquid-liquid extraction in order to cleanly extract the steroids from remaining matrix components. Prepared extracts are then analysed by LC-MS/MS in which steroids are first resolved on a reverse phase C18 column by gradient elution followed by MS/MS detection using positive atmospheric pressure chemical ionization (APCI) operated in multiple reaction monitoring mode. Chromatographic separation of several isobaric (same mass to charge ratio) steroids is essential, as is the use of deuterated internal standards for all steroids in the method.
When we applied our method to adrenal tumour samples [6], we were able to show that between 4 and 7 steroids were elevated in all ACC cases in comparison to non-ACC adrenal tumours where a maximum of 1–2 steroids were abnormal. The cortisol precursor 11-deoxycortisol was most useful in the discrimination between ACC and non-ACC adrenal lesions, whereas other steroids markedly elevated in ACC included 17-hydroxypregnenolone and pregnenolone. Indeed, all steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC. This validates the use of a panelling approach when investigating adrenal masses.
Our findings compare well with urine steroid profiling studies. Although urine steroid profiling using 24-hour collections may offer greater clinical sensitivity compared to a single blood measurement owing to diurnal rhythms of steroid production, urine measurements rely on accurately timed collections that are often performed incorrectly and are inconvenient to the patient. Advantages of our LC-MS/MS serum panel compared to urine steroid profiling by GC-MS include a less labour intensive sample preparation, as well as less expertise required for the interpretation of complex profiles, as the serum method only targets selected steroids rather than the large number of their metabolites in urine.
Use of our LC-MS/MS serum steroid panel in ACC patients has further demonstrated the limitations of assessing serum steroids by immunoassay. We observed evidence of notable interference in ACC patients in the cortisol, progesterone, 17-hydroxyprogesterone and androstenedione immunoassays, inferred to be due to elevated concentrations of structurally related steroid precursors.
Future work
Currently, our 13-steroid serum panel has been used to study a relatively small number of ACC patients (because of the rarity of the disease), and clearly larger prospective studies are required to more fully determine the diagnostic utility of our panel in ACC. Further work is also required to clarify the effects of age, sex and diurnal variation on serum steroid panelling; nonetheless the most useful markers of ACC are markedly elevated above variation attributable to these biological factors. In addition to the complexity of interpreting biomarker panels, it is not only important to consider specific reference ranges, but to also consider the patterns in results which require an omics-based analysis approach to interpretation. The challenge surrounding this, as well as the requirement for clear presentation and reporting of results to clinicians requires close involvement of clinical colleagues for the development and introduction of such testing strategies.
The analysis of steroid panels by LC-MS/MS can also undoubtedly be used in other conditions including inborn errors of steroid metabolism such as congenital adrenal hyperplasia (CAH) and polycystic ovarian syndrome (PCOS).
Although we have demonstrated the advantages of our LC-MS/MS steroid panel compared to routine immunoassays, there are undoubtedly disadvantages of using LC-MS/MS. These include the initial cost of instrument purchase, the increased expertise required and often a more laborious sample preparation. Additionally, the specificity of mass spectrometry should not be readily assumed; careful selection of multiple reaction monitoring (MRM) transitions and chromatography conditions are essential to separate isobaric steroids and other interfering compounds. However, in the context of improving the biochemical tools available to us to aid the diagnosis of ACC, the advantages of LC-MS/MS far outweigh these limitations.
Summary
In summary, LC-MS/MS serum steroid panelling offers an additional tool for the challenge that is the diagnosis of ACC. Our method combines measurement of both common and rarely measured steroids in a single sample, which we have shown provides useful data to aid the discrimination of ACC from benign adrenal tumours. Use of LC-MS/MS gives several advantages over the immunoassay and GC-MS-based methods currently used to assess steroid overproduction, but further work is required to demonstrate the full potential of its use in the diagnosis of ACC.
References
1. Fassnacht M, Kroiss M, Allolio B. Update in adrenocortical carcinoma. J Clin Endocrinol Metab 2013; 98: 4551–4564.
2. Else T, Kim AC, Sabolch A, Ramond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ, Hammer GD. Adrenocortical carcinoma. Endocr Rev 2014; 35: 282–326.
3. Arlt W, Biehl M, Taylor AE, Hahner S, Libé R, Hughes BA, Schneider P, Smith DJ, Stiekema H, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumours. J Clin Endocrinol Metab 2011; 96: 3775–3784.
4. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, et al. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med 2012;366:2189–2197.
5. Hamrahian AH, Ioachimescu AG, Remer EM, Motta-Ramirez G, Bogabathina H, Levin HS, Reddy S, Gill IS, Siperstein A, Bravo EL. Clinical utility of noncontrast computed tomography attenuation value (Hounsfield units) to differentiate adrenal adenomas/hyperplasias from nonadenomas: Cleveland Clinical experience. J Clin Endocrinol Metab 2005; 90: 871–877.
6. Taylor DR, Ghataore L, Couchman L, Vincent RP, Whitelaw B, Lewis D, Diaz-Cano S, Galata G, Schulte KM, et al. A 13-steroid serum panel based on LC-MS/MS: use in detection of adrenocortical carcinoma. Clin Chem 2017; 63: 1836–1846.
The authors
Victoria Treasure* MSc and Dr David Taylor PhD
Department of Clinical Biochemistry
(Viapath), King’s College Hospital NHS Foundation Trust, London, UK
*Corresponding author
E-mail: Victoria.treasure@nhs.net
The increasing global burden of sepsis in healthcare calls for better diagnostic tests that allow earlier detection of sepsis and infections that could lead to sepsis. The major problem for patients at risk for sepsis is an immunological imbalance. Cells of the innate immune system, such as monocytes and neutrophils, are the first-line of defence against infections. In the presence of sepsis, these cells produce a flood of inflammatory cytokines, causing widespread inflammation that can lead to death. Monocytes perform multiple immunological functions, and play a role in the development of sepsis-induced inflammation and immunosuppression. Monocyte subpopulations with different functions and morphologies vary in number over the course of the inflammatory response. The monocyte distribution width (MDW) is a novel cellular marker of monocyte anisocytosis that can add significant value to the white blood cell (WBC) count and help detect sepsis in patients entering the emergency department (ED).
by Elena A. Sukhacheva
Sepsis epidemiology and definitions
Sepsis is a major healthcare burden and, despite progress in diagnostic and treatment options, mortality from sepsis remains unacceptably high. The number of septic patients in the U.S., UK and EU is increasing [1–4]. Clearly, there is an unmet need for better diagnostic tests that can provide both the early detection of sepsis and the detection of severe infections that may progress to sepsis, if not diagnosed early enough. Global increases in sepsis frequency may be related to the aging population, as the incidence of sepsis is disproportionately increased in elderly adults, and age is an independent predictor of mortality [5]. Furthermore, immunosuppressive drugs, which are increasingly being used for diverse conditions, may result in more severe infections and increased sepsis frequency [6].
The definition of sepsis has recently been changed from the previous Sepsis-2 definition of a systemic inflammatory response (SIRS) in the presence of an infection [7], to the current Sepsis-3 definition of a life-threatening organ dysfunction caused by a dysregulated host response to infection [8]. The new Sepsis-3 definition reflects newfound understanding that the immune response in sepsis is more complex than previously thought, comprising both pro- and anti-inflammatory mechanisms.
Immune response in sepsis
It is now clear that the major problem for patients with sepsis, or at high risk of developing sepsis, is immunological imbalance, and dysregulation of the mechanisms of innate and adaptive immunity. Sepsis occurs when the immune system begins, in one way or another, to lose the battle against severe infection. After sepsis onset, the production of pro-inflammatory cytokines (IL-1β, IL-6, and tumour necrosis factor [TNFα]) by the cells of the innate immune system (neutrophils and monocytes) may result in a “cytokine storm” that produces overwhelming inflammation, which can lead to blood pressure collapse, coagulation abnormalities and, ultimately, organ failure and death. In the later stages of disease, patients who survive the cytokine storm may die from sepsis-related immunosuppression and an inability of the immune system to combat infection efficiently [9]. Inflammatory and immunosuppressive processes may overlap in sepsis [10,11], further complicating the biology of this fatal condition whose mechanisms are still poorly understood by scientists. Figure 1 shows the current understanding of immune imbalance in sepsis [12]. While all immune cells are involved in the immune response in sepsis [13–16] (Figure 2), this document is mainly focused on changes in monocytes, with other cell populations discussed only briefly.
Under normal conditions, neutrophils usually stay in the circulation for only a few hours and undergo apoptosis within 24 hours of release from the bone marrow. In sepsis, the delay in neutrophil apoptosis [17,18], combined with the increased neutrophil production in the bone marrow, results in neutrophilia. The function of these neutrophils, however, is impaired [19], with decreased chemotactic activity [20,21], decreased antibacterial function and increased production of anti-inflammatory cytokine interleukin 10 (IL-10) [22].
Sepsis also has a profound effect on all the main lymphocyte subpopulations [14]: CD4+ T-cells, CD8+ T-cells and B-cells undergo increased apoptosis; T-regulatory cells are more resistant to sepsis-induced apoptosis, leading to an increased proportion of T-regulatory cells and an immunosupressive phenotype. T-helper cell polarization from a pro-inflammatory Th1 phenotype towards an anti-inflammatory Th2 phenotype also contributes to increased immunosuppression in sepsis.
Monocytes also undergo multiple changes in sepsis, but before discussing these phenomena, it is important to discuss some basic information about the biology and classification of monocytes.
Monocytes’ biology and classification
Monocytes are cells of the innate immune system, the body’s first-line of defence against infection. Other cells of this system include neutrophils, basophils, eosinophils, mast cells, as well as certain types of lymphocytes such as γδ-T-cells and natural killer cells. The innate immune response develops during the first hours and days after pathogen invasion, and the majority of pathogens entering the human body usually are inactivated by this response and do not require adaptive mechanisms with lymphocyte involvement.
Myeloid precursors in the bone marrow differentiate into promonocytes and then into mature monocytes that enter the peripheral blood. These monocytes stay in the circulation for one to three days, after which they migrate into tissues and organs, where they turn into macrophages and dendritic cells. Morphologically, monocytes are large cells measuring 10 to 18 µm in diameter, with convoluted nuclei and azurophilic granules in their cytoplasm.
Monocytes and dendritic cells perform multiple immunological functions that include phagocytosis, antigen presentation and cytokine production. The function of these cells is regulated by a number of cell surface receptors:
Three subpopulations of monocytes have been characterized in peripheral blood [23–25]. Classical monocytes make up the main monocyte population. Expressing high level CD14 and no CD16 (CD14++CD16-), they represent 80–90% of monocytes in peripheral blood. “Intermediate” monocytes expressing CD16 (CD14++CD16+) are normally found at low numbers, but increase with cytokine stimulation and inflammation. Nonclassical monocytes display decreased expression of CD14 and increased expression of CD16 (CD14+CD16++), and comprise 9%+/-5% of all monocytes, with an average count in healthy donors of approximately 45+/-22 cells/µL [26].
In the literature, nonclassical monocytes are sometimes referred to as inflammatory or pro-inflammatory monocytes; however, published recommendations for the nomenclature of monocytes and dendritic cells in the blood clearly advocate avoiding functional terminology, “because this leads to confusion as the label ‘inflammatory’ has been used for different subpopulations in humans and mice [24].” Also, “these terms may prematurely ascribe functional attributes to cells based on ex vivo studies while they largely remain to be functionally characterized in vivo [24].” Subsets of nonclassical monocytes are expanded dramatically in several pathological conditions including sepsis [26–28], HIV-1 infection [29–33], diabetes [34–35], tuberculosis [36] and other disease states [37].
The recent detailed analysis performed by Mukherjee et al. [28] revealed the functions of monocyte subsets as follows: classical monocytes are phagocytic with no inflammatory attributes, nonclassical subtypes display inflammatory characteristics on activation and display properties for antigen presentation, and intermediate subtypes appear to have both phagocytic and inflammatory functions [28]. In 2017, research based on single-cell RNA sequencing discovered even more subtypes, describing six subpopulations of dendritic cells and four monocyte subpopulations [39]. This classification was based solely on transcriptional activity, and further studies will be needed to understand function and describe the phenotype of all cell subpopulations. Nonetheless, it is clear that morphologically similar cells that we call monocytes may actually have very different functions in human immunity.
Monocytes in sepsis
Monocytes, as cells of first-line defence against infection, are involved in the immune response from very early stages. Abundant literature exists on monocytes and the changes they undergo in sepsis.
A recent study on the dynamics of monocyte subpopulations in peripheral blood at the onset of infection has demonstrated a decrease in the number of peripheral blood monocytes during the early stages of lipopolysaccharide (LPS)-induced acute inflammation in humans. This loss may be due to the migration of monocytes from the blood into tissues, where they differentiate into macrophages and dendritic cells, or it may reflect an increase in monocytes residing in the marginal pool or rolling on the vessel walls [40]. For all three subpopulations of monocytes, the number of cells was decreased at one to two hours after LPS injection. This decrease was followed by a return to the baseline count, but with differences in timing for the three monocyte subsets. This difference in timing means that the early stages of infection, before the appearance of any clinical symptoms, are characterized by differences in the proportions of monocyte subpopulations relative to baseline pre-infection proportions.
Functional changes in monocytes and, in parallel, changes in their cellular morphology, have been demonstrated in the past for a human THP-1 monocytic cell line infected with viable C. pneumonia bacteria [41]. The differentiation of infected cells into macrophages was accompanied by a change to an ameboidor diffused morphology as assessed by microscopy after Giemsa staining.
Multiple studies have demonstrated the importance of HLA-DR expression on monocytes as a prognostic marker in septic patients. A decreased level of HLA-DR expression on monocytes has been found to be a negative prognostic indicator [42–44] and may be used to evaluate the functional activity of the immune system [45,46]. Decreased HLA-DR, as a marker of monocyte anergy, correlates with decreased antigen presentation capacity and decreased pro-inflammatory cytokine release. This has been analyzed mainly by flow cytometry, but, recently, new methods based on real-time PCR have emerged [47,48].
Another monocyte marker, CD16, plays an important role in orchestrating the response of monocytes to Gram-negative sepsis. It has been demonstrated that CD16 on human monocytes is a key regulator of the TRIF-dependent TLR4 signalling pathway, and this pathway is preferentially activated in the CD16+ monocyte subset [49]. Recent publications suggest the variability of monocyte properties in sepsis. Detailed analysis of gene expression in patient monocytes during sepsis and after recovery demonstrated plasticity of monocytes in the course of disease [50]. The significant up-regulation of pro-inflammatory cytokines (IL-1b, IL-6) and chemokines (CCL3 and CCL5) has been demonstrated in sepsis monocytes compared to monocytes after recovery. Transcriptional factor NF-kB, a central transcriptional regulator of the inflammatory response, was also activated in sepsis monocytes, supporting their involvement in severe inflammation. At the same time, anti-inflammatory cytokine IL-10 was found to be up-regulated in sepsis monocytes. These studies once again highlight the diversity of monocytes’ function in sepsis pathogenesis, and their key role in disease progression, with the possible polarization from a pro-inflammatory state to an immunosuppressive state.
More recently, Crouser et al. demonstrated that the morphological variability that occurs during monocyte activation in the early inflammatory response can be captured by measuring the monocyte distribution width (MDW), an indicator of monocyte anisocytosis. Investigators showed that MDW could be a novel cellular marker that may help detect sepsis early in patients admitted to the emergency department (ED) [51]. Multiple morphometric characteristics of monocytes were obtained using a DxH 800 cellular analysis system, which employs physical measurement of cell volume, conductivity and multiple angles of laser scatter to classify leukocytes into five sub-populations and detect the presence of abnormal cells. This study showed that anisocytosis of circulating monocytes provides significant added value to WBC count for the detection of sepsis in the ED population.
Conclusion
In summary, monocytes are a very heterogeneous population of cells that differ in phenotype, size, nuclear morphology, gene profile and function [52]. In sepsis, this diversity is even more pronounced due to functional changes of monocyte subsets, and is accompanied by a variation in monocyte morphology.
Morphological variability is just the tip of the iceberg of the underlying biological heterogeneity, and may be an important early marker of sepsis or severe infections with a high risk of progressing to sepsis. A recent publication from Crouser [51], together with previous research on sepsis using cellular morphometric parameters gathered using a DxH 800 analyser [53–56], may build the foundation for practical usage of MDW in combination with currently-used sepsis markers (WBC, PCT, CRP, IL-6) for early sepsis screening and diagnosis, leading to early initiation of appropriate therapy.
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The author
Elena A. Sukhacheva, Ph.D.
Senior Manager,
Global Scientific Affairs, Hematology, Beckman Coulter Diagnostics,
Miami, FL, USA
March 2026
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