A mini review of the assessment of vitamin E status in patients with familial hypobetalipoproteinaemia

Introduction

Familial hypobetalipoproteinaemia (FHBL) subtypes secretory deficit (SD) 1, 2 and 3, due to homozygous loss of function mutations in the MTTP, APOB and SAR1B genes respectively, are characterised clinically, in part, by deficiencies of the fat soluble vitamins (1). The neurological and ophthalmological complications, namely progressive sensorimotor neuropathy, ataxia and retinitis (1-3) may be slowed or halted by supplementation of vitamins A and E (1,2). These subtypes of FHBL are often life-limiting if not treated, with the neuropathy possibly progressing to the point of causing respiratory failure by the third decade without intervention (2).

However, one significant issue arises when trying to monitor the effectiveness of Vitamin E supplementation. Vitamin E, primarily in the form of α-tocopherol in humans, lacks a dedicated carrier protein in circulation, and instead is transported within lipoproteins (1,4). Patients with FHBL SD 1, 2 and 3 have little to no circulating Apolipoprotein B containing lipoproteins (1). Therefore, serum or plasma levels of vitamin E will always appear low in patients with these conditions. Vitamins A and D do not suffer this drawback and serum levels can be used appropriately for monitoring (1). The adequacy of vitamin K supplementation can be assessed using clotting studies, as prolonged prothrombin time and activated partial thromboplastin time will signal deficiency (1).

The importance of vitamin E deficiency in the development of serious neurologic sequelae is borne out by development of similar complications in other causes of actual or functional vitamin E deficiency, such as inherited defects in liver transfer of Vitamin E into lipoproteins in another condition called ataxia with isolated vitamin E deficiency (5,6). Vitamin E concentrations in serum are often reported as a ratio to cholesterol to try to correct for its close correlation with lipoproteins. However, in patients with FHBL SD 1, 2 or 3 this ratio is likely invalid (7). Very low cholesterol levels in circulation may create ratios that vary widely but do nothing to inform the adequacy of vitamin E delivery to nervous tissue, the primary concern in FHBL. Notably, over 90% of total body α tocopherol in mammals is in the muscle, liver and adipose tissue (8). Ideally, clinicians looking after patients with FHBL should be able to get real-time feedback on the adequacy of vitamin E supplementation to adjust doses as needed. Since this is not available, standard practice is to give very large amounts of vitamin E (1,2). This difficulty in the assessment of vitamin E stores using plasma levels may also be of some relevance in patients with other causes of very low lipoproteins, including the use of lipid lowering medications.

This mini review will explore alternative ways of monitoring vitamin E status in patients with FHBL as published in the literature. This is of importance as the current management paradigm when it comes to vitamin E supplementation, without an accurate and reliable biomarker for monitoring, may be resulting in harm to some patients with FHBL due to either over or under dosing. The limitation of the commonly used measurement of serum or plasma vitamin E is already well known in these patients as we have highlighted. What is needed for patients living with FHBL SD is a better biomarker. Our mini review will describe what is known about the currently available alternative assessment methods. However, this field is one that we believe requires a lot more research.

Mini review findings

Metabolites

There have been attempts to measure vitamin E metabolites in blood. Bartolini et al. measured multiple metabolites (9). These included α-tocopherol, γ-tocopherol, long-chain metabolites (LCMs) α-13'hydroxychromanol, 13'-carboxychromanol and the free radical-derived LCM α-tocopheryl quinone, the medium-chain metabolite (MCM) 2,7,8-trimethyl2-(δ-carboxymethylbutyl)-6-hydroxychroman (α-CMBHC) and the short-chain metabolites (SCMs) α- and γ-carboxy-ethyl-hydroxychroman metabolites as well as metabolites not fully clarified but called M1, M2 and M3. These metabolites were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). They also measured, by Western blot, proteins whose expression is modulated by vitamin E, namely the pregnane X receptor and cytochrome P450 subtype 4F2. The investigators measured these before and after a week of α-tocopherol supplementation in 17 healthy volunteers. They found that the biomarkers did change with supplementation, but there was great variation in the response among the volunteers. Only α-carboxy-ethyl-hydroxychroman, α-13'hydroxychromanol and M1 had correlations with supplementation as well as low enough inter-individual variation among the volunteers to be considered possible candidates for monitoring vitamin E status. However, all seemed to be significantly impacted by cholesterol concentration in plasma and therefore none were vastly superior to measurement of α-tocopherol (9).

No data could be found in the literature for how these metabolites correlated with tissue stores of vitamin E. The finding by Bartolini et al., that cholesterol concentration in blood continued to strongly influence metabolite levels suggests that these measurements are subject to the same limitation seen when assessing α-tocopherol directly (9).

Adipose tissue

As a fat-soluble vitamin, vitamin E has a large adipose depot (8,10,11). Handelman et al. showed that during yearlong supplementation of four healthy adults with all-rac-α-tocopherol, the ratio of α:ϒ tocopherol in adipose taken at biopsy from the gluteal region increased linearly followed by a linear decrease after cessation (12). During the year of supplementation adipose biopsies were taken five times, at least 2 months apart and again for the year following cessation of the supplements. During this time, the α tocopherol to cholesterol ratio in the adipose biopsies, which the authors used as the measure of α tocopherol concentration in the adipose, did not always change. In fact, the linear increase and then decrease in α:ϒ tocopherol ratio was driven largely by linear decrease and then increase in ϒ tocopherol with supplementation and cessation of supplementation respectively. The conclusion made from this finding is that adipose stores and releases α tocopherol very slowly. Thus, it may not be a good store to protect against deficiency, as release of α tocopherol would be too slow to supply other tissues that need it. ϒ tocopherol changed more quickly (12).

The study also included a cross-sectional component in which five long-term α tocopherol supplement users and five non-users each underwent a single adipose tissue biopsy; with those having taken supplements exhibiting significantly higher α:γ-tocopherol ratios (12). ϒ tocopherol, the predominant vitamer of vitamin E from plant sources, does have biological activity in humans, though less than α tocopherol (13). The plasma concentration of ϒ tocopherol is usually much lower than that of α tocopherol (14). Other studies have shown that supplementation with α tocopherol causes a reduction in plasma ϒ tocopherol (15,16). A similar thing appears to be happening here in the adipose.

Kayden et al. published a study comparing the adipose vitamin E content of surgically acquired specimens from 4 patients undergoing surgery, none of whom had FHBL, with vitamin E content of adipose taken by needle biopsy from the gluteal region of 14 other patients (17). Ten of the latter had FHBL-SD. Intake of supplemental vitamin E tended to result in higher tocopherol to triglyceride ratios in the adipose samples, but the authors noted there was no simple dose to tissue correlation. Lack of supplementation in patients with FHBL-SD resulted in low values, but some patients with the condition who took very high doses of vitamin E also had low values (17). This study did successfully show that vitamin E content of adipose could be reproducibly measured in samples acquired by needle biopsy, potentially making the use of adipose more accessible compared with surgical acquisition of specimens (17). However, what it could not answer was whether the lack of correlation of levels measured in adipose with reported supplementation was due to inadequate supplementation doses or duration or due to the disease process itself. MTTP, the gene defective in FHBL SD1, is expressed in adipocytes and may impair the movement of tocopherols into and out of the cells if defective.

Handelman et al. further confirmed the reproducibility of biopsies obtained using needles (18). Schäfer and Overvad performed needle biopsies on healthy persons and found that vitamin E content correlated well with intake (19).

Cuerq et al. conversely, showed that adipose tissue vitamin E status did not change significantly after 4 months of supplementation with 50 IU/kg/day of either α-tocopherol or a water-soluble derivative called tocofersolan in three patients with FHBL SD1 or in four patients with FHBL SD3 (20). They hypothesised that this might have been different if larger doses or a longer period was used (20). They did find that the FHBL SD3 patients started with higher adipose α-tocopherol per gram of adipose than the FHBL SD1 patients (20).

The repeatedly demonstrated slowness of change in adipose α tocopherol in response to changes in supplementation, along with the invasiveness of obtaining a sample, may limit its use in routine monitoring of patients with FHBL.

The challenges seen in use of adipose for assessment of vitamin E status are shown in Table 1.

Blood cells

It will be appreciated that repeated fat biopsies in patients with FHBL-SD who need life-long follow up may be impractical, especially considering that diagnosis is often made in childhood. Biomarkers of vitamin E status from blood samples would therefore be more pragmatic and there have been studies exploring measurement of tocopherols in blood cells.

Lehmann et al. gave ten healthy men and ten healthy women α tocopherol at 30 mg daily for six weeks and then 100 mg daily for a further 6 weeks (21). Two men and two women were given placebo. Fasting venous blood was collected at baseline, 6 weeks, and 12 weeks. Tocopherols were measured using high performance liquid chromatography (HPLC) with fluorescence detection after extraction with chloroform-methanol for red cells and lymphocytes and chloroform-methanol-pyrogallol for platelets. Plasma tocopherol and lipids were also measured. Platelet α tocopherol appeared to show the best correlation with intake with least interference by plasma lipid concentrations, followed by lymphocytes. Plasma and red cell α tocopherol were both significantly correlated with plasma lipid concentrations (21).

Kayden et al. described an alternative method of measurement of red cell tocopherol content. They used thin-layer chromatography on silica plates (22). The tocopherol band was identified using ultra violet (UV) light, then scraped into a centrifuge tube, eluted from the silica using ethanol, then centrifuged at 25,000 rpm. The ethanol solution then underwent spectrophotometric quantification after addition of bathophenanthroline, FeCl₃ and H₃PO₄ (22).

More recently, Steghens et al. described a reversed-phase HPLC with diode array detection for measurement of vitamin E, among other metabolites (23). This was the method used by Cuerq et al. for measurement of plasma, red cell and adipose α tocopherol (24) and most modern methods utilise HPLC with either diode array detection or tandem mass spectrometry (14).

Nordoy et Strom had shown that transfer of tocopherols from lipoproteins into red cells occurs faster than from lipoproteins into platelets (25). Vatassery et al. found that in 49 healthy males aged 24 to 91 years old, platelet tocopherol did not vary with lipid concentrations in blood while red cell tocopherol did (26). Clarke et al. came to a similar conclusion, namely that platelet tocopherol analysis was a better measurement of intake than either plasma or red cell assessment due to how dependent the latter two matrices were on the circulating lipoprotein concentration (27).

However, there have been other studies that showed that red cell concentration in FHBL can improve, in some cases even to normal levels, with supplementation of vitamin E, despite there still not being enough lipoproteins present to allow for normalisation of the plasma/serum vitamin E. Di Filippo et al. published a case report of a child with FHBL-SD1 who showed exactly this (28). Similarly, the study by Cuerq et al. described earlier in this section, in which four patients with FHBL SD1 were included, showed that four months of supplementation with 50 mg/kg/day of tocopherol acetate was able to bring their red cell α tocopherol from low to normal, while again their plasma vitamin E levels did not normalise (24). In this study, adipose tissue vitamin E levels did not change after the four months of supplementation (24). This work was very helpful in that it also included 166 healthy children from whom the authors created reference intervals for α tocopherol in plasma (11.9–30 µmol/L), red cells (2.0–7.8 µmol/L packed red cells) and adipose (60–573 nmol/g of adipose) (24).

Yaoi et al. published in 1984 an attempt to create reference intervals for platelet α tocopherol for adults and children (29). They studied 29 adults and 17 children who were apparently healthy. They found that in the adults there was a strong correlation between the platelet tocopherol measurement and the ratio of plasma tocopherol to lipids and also a strong correlation between platelet and red cell tocopherol (29). However, in the children they found that neither platelet nor red cell tocopherol corresponded well with the ratio of plasma tocopherol to plasma lipids. The reason for this difference between adults and children was not clear (29).

The advantages as well as potential drawbacks of use of red cells and platelets are summarised in Table 1.

Discussion

The monitoring of vitamin E status in patients with FHBL SD is not straightforward. Due to the limitations inherent in the plasma or serum measurement caused by the significant dependence on lipoproteins for blood transport, adipose and blood cells have been studied as alternative matrices for measurement. However, these also pose some challenges as we have seen. It has been shown in studies of serial measurements that adipose α-tocopherol increases very slowly, if at all, with supplementation both in healthy individuals and in those with FHBL SD (12,20). In patients with FHBL-SD, adipose levels did not correlate well with reported supplementation amounts, except that those who did not take any supplementation at all had very low levels (17).

Both platelets and erythrocytes have shown potential for use as more reliable vitamin E measurement matrices (21,22,24-26,29). Arguments for platelet use as superior to red cells have been made citing experiments that showed red cell α tocopherol concentration can be strongly influenced by lipid concentration (21,25-27), suggesting that the same issue that limits the measurement of vitamin E in plasma or serum would make red cell estimates unreliable as a guide to whole body stores in patients with FHBL SD. However, it was demonstrated that, at least in patients with FHBL SD3, red cell α tocopherol can be increased with oral supplementation to levels seen in healthy children without changing their circulating lipoprotein concentrations (24). It can also be postulated that with the longer circulating survival time of red cells (120 days) versus that of platelets (10 days), red cells would have had more exposure and thus may be more representative of longer-term vitamin E intake than platelets.

Cuerq et al. created reference ranges for healthy children for plasma, adipose and red cell α tocopherol (24). The goal of this effort was to be able to use these markers for the monitoring of children with FHBL-SD3.

As is often the case with rare conditions, progress has been slow in delineating the best way to monitor vitamin E status in patients with FHBL SD. This is illustrated by the fact that much of the published literature we have found and reviewed here is over 40 years old. Understandably, the patient cohorts are small and studies investigating the best way forward can be difficult to recruit for. Simple extrapolation from healthy subjects poses the risk of overlooking potential disease specific parameters, such as the presence of the MTTP gene product in adipose that may make adipose tocopherol kinetics different between those with and without FHBL SD1. More importantly, the main clinical outcome, namely what level of α tocopherol in adipose or red cells or platelets leads to the halting of progression of neurological sequelae of vitamin E deficiency in these patients with FHBL SD, will be exceedingly difficult to ascertain. This, however, is probably the most important question for future research to answer if we are going to manage these patients safely. Again, numbers will be very small and the time needed for such outcome studies will be long, given the natural history of the diseases. For now, we may have to assume that normalisation of vitamin E in these matrices is desirable and adequate for this goal, but given the biochemical complexities involved, we cannot be certain of this.

More work in the area of vitamin E status assessment for this group of patients is sorely needed. The current practice in many areas of blindly giving patients with FHBL SD very high doses of vitamin E may run the risk of undertreating some. It is accepted that many patients have progression despite high dose replacement (2). This may be because of the large pill burden leading to reduced adherence (1) or because those patients with progression still have poor body stores despite the high dose and would actually require even higher doses. Additionally, high doses of vitamin E may not be entirely benign. High doses of this vitamin can impair function of vitamin K, which may worsen coagulopathy in these patients (30).

Improvement of accuracy of vitamin E status assessment with alternatives such as circulating blood cells or adipose, if they become clinically embedded, may lead to better assessments for other persons without FHBL SD.

Conclusions

The currently widely used methods of measuring vitamin E in serum or plasma and correcting for plasma lipid concentrations are inadequate for accurate assessment of vitamin E status in patients with SD subtypes of FHBL. Alternative assessment matrices from published literature, namely measurement in adipose tissue, platelets or red cells, appear to hold promise for improving this, but are currently not prevalent in routine clinical use and do appear to have some limitations. Further work in their development and validation, and outcome studies showing their impact on the management of patients are needed.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Journal of Laboratory and Precision Medicine for the series “Adult Inherited Metabolic Disorders”. The article has undergone external peer review.

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0009/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0009/coif). The series “Adult Inherited Metabolic Disorders” was commissioned by the editorial office without any funding or sponsorship. N.L. and A.M. served as the unpaid Guest Editors of the series. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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