Homocysteine, vitamin B₁₂, folates, vitamin B₆, choline, betaine

13.1 Hyperhomocysteinemia and degenerative diseases

Wolfgang Herrmann, Rima Obeid

13.1.1 Introduction

Homocysteine is a non proteinogenic, sulfur-containing amino acid of the methionine metabolism. Only 1–2% of homocysteine circulate freely in the blood in the reduced form, 70–90% of plasma homocysteine are protein-bound and the remaining proportions are disulfides, such as homocystine or the mixed disulfide homocysteine-cysteine (Fig. 13.1-1 – Homocysteine forms).

Homocystinuria is a congenital disorder of amino acid metabolism. This metabolic disorder is associated with a strong increase in plasma homocysteine concentration and markedly increased urinary excretion of the oxidation product homocystin /1/. If untreated, affected individuals develop already in their youth severe atherosclerotic vascular alterations, mental retardation and bone deformation besides other symptoms. According to the homocysteine theory of atherosclerosis, there is a causal association between significantly increased plasma homocysteine concentrations and atherosclerosis /1/. Numerous studies have provided evidence that even moderately elevated plasma homocysteine concentrations are positively associated with an increased risk of cardiovascular disease, venous thrombosis, peripheral arterial occlusion disease and stroke.

Hyperhomocysteinemia is considered as an independent risk factor for cardiovascular disease and thought to account for about 10% of the total risk /2, 3/. Hyperhomocysteinemia is also a risk factor for neurodegenerative diseases such as vascular dementia, Alzheimer’s dementia or cognitive impairment /4/. Older adults, in particular, have a high prevalence of hyperhomocysteinemia due to common vitamin deficiencies /5/ that can easily be treated with adequate vitamin substitution.

13.1.2 Homocysteine metabolism

Homocysteine stands at the intersection of two metabolic pathways: catabolic transsulfuration and the remethylation cycle (Fig. 13.1-2 – Homocysteine metabolism) /6/. Homocysteine is either transsulfurated to cystathionine or remethylated to methionine. The remethylation rate depends on the dietary methionine intake. During remethylation, homocysteine binds a methyl group of 5-methyltetrahydrofolate (5-methyl-THF) and forms methionine. The reaction occurs in all tissues and is catalyzed by the vitamin B₁₂-dependent enzyme methionine synthase (EC 2.1.1.13).

Alternatively, the methyl group can also be provided by betaine. However, this reaction is present only in liver and kidney. Transfer of the methyl group from betaine to homocysteine is catalyzed by the enzyme betaine homocysteine methyltransferase (BHMT, EC 2.1.1.5) /7/.

The product of betaine de methylation is dimethylglycine (DMG), which can be further converted to sarcosine and later to glycine. Betaine is formed by enzymatic oxidation of choline. Consequently, choline deficiency can be a cause of hyperhomocysteinemia /8/.

Methionine is converted to S-adenosylmethionine (SAM), which functions as a universal donor of methyl groups.

The de methylation of SAM results in the formation of S-adenosylhomocysteine (SAH), from which homocysteine is released by hydrolytic cleavage. SAH is a potent competitor for binding sites of SAM on methyltransferases and, therefore, can inhibit transmethylation /9/.

In the catabolic transsulfuration pathway, homocysteine condenses with serine to form cystathionine. This reaction is catalyzed by cystathionine-β-synthase (CBS, EC 4.4.1.1), a vitamin B₆-dependent enzyme.

In a second reaction step catalyzed by γ-cystathionase (EC 4.4.1.1), likewise a vitamin B₆-dependent enzyme, cystathionine is hydrolyzed to form cysteine and α-keto butyrate. SAM plays an important role in the regulation between the metabolic pathways of remethylation and transsulfuration /10/.

SAM activates the CBS and inhibits the synthesis of 5-methyl-THF catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20).

The remethylation activity catalyzed by glycine N-methyl transferase (GNMT, EC 2.1.1.20) plays an important role in the regulation of SAM consumption, where the methyl group is transferred from SAM to glycine. The reaction is inhibited by 5-methyl-THF.

13.1.3 Hyperhomocysteinemia

As homocysteine is highly cytotoxic, its intracellular concentration is kept low by remethylation to form methionine and by a cellular homocysteine export mechanism to the plasma /11/. Most of the plasma homocysteine (about 70%) is metabolized renally (remethylated) and only a small proportion is excreted unchanged in urine.

Moderate hyperhomocysteinemia with plasma concentrations of 12–30 μmol/L can have various causes:

• Deficiencies in folate, vitamin B₁₂ or vitamin B₆
• Enzyme deficiency, the best known being MTHFR deficiency
• Chronic kidney disease /12/.

Refer to Tab. 13.1-1 – Homocyteine level in fasting plasma and following oral methionine load.

Plasma homocysteine levels above 30 μmol/L are primarily caused by genetic enzyme deficiencies or renal dysfunction. Homocysteine metabolism is influenced by numerous substances, drugs, diseases and life-style factors, mostly as direct or indirect antagonists of co factors and enzyme activities, but also by disulfide exchange reactions, resorption disorders and enzyme induction (Tab. 13.1-2 – Causes of homocysteine changes and Tab. 13.1-3 – Mutations associated with hyperhomocysteinemia).

13.1.3.1 Genetic influences

Homocysteine levels between 30–100 μmol/L are primarily caused by heterozygous enzyme defects. Severe hyperhomocysteinemia with concentrations above 100 μmol/L is likely caused by an enzyme defect with a marked loss of enzyme activity (Tab. 13.1-3 – Mutations associated with hyperhomocysteinemia).

13.1.3.2 Associated diseases

Diseases associated with hyperhomocysteinemia are atherosclerosis, cardiovascular disease, congestive heart failure, thrombosis, stroke, neural tube defects, pregnancy complications, cognitive dysfunctions, depression, vascular dementia, Alzheimer’s dementia, hypertension and tumors.

Diseases, drugs, and conditions associated with hyperhomocysteinemia are described in Tab. 13.1-4 – Causes and diseases possibly associated with hyperhomocysteinemia.

13.1.3.3 Age and gender dependency

Homocysteine concentration generally increases with age; in younger age, men usually have higher levels than women. In about 40-year-olds, the gender-related difference is about 2 μmol/L and can be explained with the estrogen effect in women. The gender-related difference decreases rapidly during menopause.

Age-dependent increase in homocysteine is, at least partly, explained with the physiological decline in renal function /5/. The increase in homocysteine concentration is linear up to 60–65 years of age and then becomes much faster, increasing by about 10% or 1 μmol/L per decade on average /3/.

13.1.4 Homocysteine reduction through folic acid supplementation

The homocysteine-lowering effect of different folic acid doses was studied /44, 45, 46, 47/. Participants with higher baseline homocysteine concentrations (16.6 μmol/L) had the greatest reductions in plasma homocysteine in response to daily folic acid doses of 0.2 mg (–20.6%), 0.4 mg (–20.7%), and 0.8 mg (–27.8%). In those with lower baseline homocysteine levels (10.1 μmol/L), the responses to the same doses were –8.2%, –8.9% and –8.3%, respectively /44/. Folic acid supplementation caused a linear, dose-dependent increase in folate concentration in blood. Due to the six-month supplementation of 200 μg/d, plasma folate was increased from 18 to 34 nmol/L and reached about 50% of the increase attained with 800 μg/d. Long-term supplementation with low folic acid doses (200 μg/d) are sufficient to efficiently improve homocysteine metabolism. Refer in addition to Fig. 13.1-5 – Median changes in homocysteine concentrations after 26 weeks of treatment with placebo or 0.2 mg, 0.4 mg or 0.8 mg folic acid per day).

13.1.5 Hyperhomocysteinemia as risk factor

Hyperhomocysteinemia is a general risk factor for many organ systems.

13.1.5.1 Vascular disease

Meta-analyses /48/ of retrospective and prospective studies emphasize the relation between hyperhomocysteinemia and degenerative vascular disease. Statistical analyses of this data regarding an increase in homocysteine of 5 μmol//L calculated an odds ratio of 1.32 for the incidence of ischemic heart disease, 1.60 for venous thrombosis and 1.59 for stroke. Moreover, these meta analyses show that lowering of the plasma homocysteine concentration by 3 μmol/L compared to the baseline level could be associated with a 16% lower risk of ischemic heart disease, 25% lower risk of thrombosis or 24% lower risk of stroke /2/. Furthermore, a prospective study over more than four years reported a significant decrease in the plaque volume of the carotid artery after supplementation with B vitamins /49/. In addition, a significant decrease of the carotid intima media thickness has been reported in patients at risk of cerebral ischemia after one-year supplementation with B vitamins /50/.

Secondary prevention studies

Despite the association between hyperhomocysteinemia and cardiovascular disease, the lowering of the disease rate by reduced homocysteine concentration following vitamin therapy as a key element of the causality chain has remained unsolved.

More than 50,000 individuals have been included in intervention studies worldwide to clarify the potential benefit of homocysteine lowering therapy with B vitamins (secondary prevention):

• NORVIT Study /51/ (3,749 patients who had had an acute myocardial infarction were additionally treated with B vitamins for three years)
• VISP Study /52/ (3,860 stroke patients were treated with low-dose or high-dose B vitamins for two years)
• HOPE-2 Study /53/ (5,522 patients who had vascular disease or diabetes were treated with B vitamins or placebo for five years)
• Search Study /54/ (12,064 survivors of myocardial infarction received 2 mg of folic acid plus 1 mg of vitamin B₁₂ and/or placebo daily for 6.7 years).

In these studies, homocysteine serum level was lowered significantly by 17–28%, but no significant risk reduction regarding the end points (myocardial infarction, stroke) was observed. The clinical significance of secondary prevention studies is clearly limited, primarily because, due to the basic medication of participating patients, it is difficult to provide evidence of the additional effect of homocysteine reduction by B vitamins on the cardiovascular end points and, as a result, the diagnostic power is only considered adequate in large-scale meta analyses.

The situation is different in stroke prevention. Here, the expected risk reduction is about 25%; hence, a meta analysis requires considerably fewer patients in order to draw a well-founded conclusion.

Meta analyses

One meta analysis /55/ comprises data from 75 studies (22,068 patients and 23,618 controls) as well as 14 randomized trials on 39,597 participants with cardiovascular disease (CVD) events and homocysteine lowering. As a result, an independent significant association between plasma homocysteine and the risk of ischemic cardiovascular disease was found. The CVD risk was 16% higher in individuals who were carriers of genotype MTHFR-677 TT than in those with genotype CC (wild type); the difference in plasma homocysteine between the genotypes TT and CC was 1.9 μmol/L. The 14 randomized trials showed no significant reduction in CVD risk, despite a reduction in homocysteine by 3.3 μmol/L. However, in studies with low-dose aspirin use, homocysteine reduction was associated with a reduction in CVD risk by 7%. The CVD risk was not reduced in studies with high-dose aspirin intake. It is therefore concluded that folic acid would have a role in the primary prevention of CVD, when aspirin is not taken routinely, but not in secondary prevention /55/. For further meta analyses refer to References /56, 57, 58/.

13.1.5.2 Neurotoxic effect

Homocysteine is able to compete with receptors for glutamate and aspartate in the central nervous system (CNS). As a result, hyperhomocysteinemia can have an excitotoxic effect on various N-methyl-D-aspartate (NMDA) glutamate receptor subtypes and, thus, contributes to an increased formation of hydroxyl radicals /59/ (Fig. 13.1-7 – Mechanisms of homocysteine neurotoxicity). The activation of NMDA receptors is thought to promote intracellular calcium accumulation and a resulting release of cellular proteases and, possibly, apoptosis. This excitotoxic mechanism has been postulated for a number of neurodegenerative and psychiatric diseases, from metabolic and toxic encephalopathy to schizophrenia. It has also been shown that both NMDA receptors and group I metabotropic glutamate receptors are involved in homocysteine-induced neurotoxicity.

13.1.5.3 Methylation capacity defect

In addition to its effect as a vessel damaging agent, hyperhomocysteinemia is also an indicator for defective methylation capacity (elevated SAH and/or low SAM). Intact interaction between folate and vitamin B₁₂, as occurs with normal homocysteine levels, is very important for DNA synthesis and various methylation reactions (methylation of DNA, RNA, myelin, phospholipids, receptors and neurotransmitters). It has been reported that patients with neuropsychiatric disorders had reduced concentrations of folate /38/ and SAM as the active methyl group donor in cerebrospinal fluid /60/. The SAM levels post-mortem were very low in the brain and also in the cerebrospinal fluid of patients with Alzheimer’s dementia.

In Alzheimer’s disease, changes in the SAM status are considered to be important for the expression of presenilin, a key factor for β-amyloid formation, and for the formation of phosphorylated tau protein) /61/.

Refer to Fig. 13.1-8 – Correlation between SAH and the SAM/SAH ratio in cerebrospinal fluid (CSF) with β-amyloid 1–42 concentration in the CSF in dementia-free patients.

The concentrations of phosphorylated tau protein in cerebrospinal fluid are positively and age-independently associated with the SAH concentration in cerebrospinal fluid (Fig. 13.1-9 – Concentrations of SAH in cerebrospinal fluid (CSF) in relation to P-tau 181 concentrations in the CSF observed in three age groups) /62/.

It is suggested that the over expression of the gene is linked to hypo methylation of the gene’s promotor region. The synthesis and catabolism of catecholamines and myelin formation can be modulated by reduced SAM concentration. Patients with depression and high plasma homocysteine had low concentrations of monoamine metabolites in the cerebrospinal fluid, which points to defective turnover of serotonin, dopamine and noradrenaline. Elevated homocysteine concentrations, but low methionine and tryptophan, the precursor of serotonin, have been reported in patients with Alzheimer’s disease.

Normal folate metabolism is also important for the synthesis of sufficient tetrahydrobiopterin, a cofactor determining the speed of serotonin and dopamine synthesis. Low vitamin B₁₂ and folate lead to decreased formation of tetrahydrobiopterin in the CNS /63/. Elevated homocysteine levels and markedly low methionine and tryptophan concentrations were found in patients with early stage of Alzheimer’s disease.

13.1.5.4 Atherothrombotic effect

The homocysteine metabolism in cardiovascular cells is limited to folate- and vitamin B₁₂-dependent remethylation. This conclusion is based on the fact that no transsulfuration has been proven in the endothelium cells of human vessels to date /64/. Because there is no irreversible degradation of homocysteine to cysteine, homocysteine synthesis can quickly exceed cellular export and cause specific cell injury up to cell death. More than any other organ, the cardiovascular system is especially sensitive to increases in homocysteine. Such increases can induce morphological changes of the vessel wall, stimulate inflammation, activate the endothelium and coagulation cascade and inhibit fibrinolysis. In general, hyperhomocysteinemia induces the impairment of antithrombotic endothelial function and creation of a pro coagulatory milieu /65/. Atherogenic mechanisms most frequently discussed in hyperhomocysteinemia include thickening of the vessel wall intima, increased platelet turnover, increased platelet activation with increased thromboxane synthesis, endothelial dysfunction, activated leukocytes, increased LDL oxidation, increased foam cell formation due to lipid deposits in the vessel wall and increased proliferation of smooth muscle cells.

The atherogenic process in hyperhomocysteinemia is mostly thought to be triggered by oxidative stress. The formation of homocysteine thiolactone, a highly reactive internal thioester, and inflammation are discussed as key mechanisms in this context.

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13.2 Homocysteine

Wolfgang Herrmann, Rima Obeid

13.2.1 Indication

Patients with atherosclerotic vascular and neurodegenerative disease.

Target groups for homocysteine determination:

• Vascular disease: coronary heart disease, myocardial infarction, atherosclerosis of the carotid artery, peripheral arterial occlusion disease, atherosclerosis of the cerebral arteries, cerebral insult, venous thrombosis, pulmonary artery embolism
• Risk groups for cardiovascular disease: positive family history, arterial hypertension, smoking, hyperlipidemia, renal insufficiency, diabetes, metabolic syndrome, adiposity
• Risk groups for vitamin deficiency: elderly, vegetarians, inflammatory gastrointestinal disease (gastritis, malabsorption), alcohol abuse, one-sides eating habits, drugs
• Other risk groups: dementia, cognitive impairment, depression, homo cystinuria, hypothyroidism, rheumatism, AIDS, pregnancy complications (preeclampsia, HELLP syndrome, neural tube defect, intrauterine growth retardation)
• Prophylaxis: preconceptional prevention of neural tube defects, healthy individuals > 50 years.

13.2.2 Method of determination

High performance liquid chromatography (HPLC)

HPLC is the standard method for determining the homocysteine level in blood plasma or serum /1/. Because of their detection limit, simplicity and high throughput capacity, the HPLC method in combination with fluorescence detection (HPLC-FD) are widely used methods for aminothiol quantification /2/. There are considerable variations between the procedures for sample processing, chromatographic conditions and sample detection which limit the standardization of the method.

The applied methods can principally be divided into procedures using derivatization and those working without derivatization. The first procedure uses pre-column derivatization with fluorogenic reagents reacting with SH groups. Three derivatization reagents are commonly used: monobromobimane, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole 4-sulfonate being the most suited. Electrochemical detection, which does not use sample derivatization, is attractive because of very easy sample processing, high methodical specificity, the possibility of auto injection with high sample throughput and the possibility of co-determining other thiols.

This procedure has the disadvantage that the flow cell and electrodes become easily contaminated. Amperometric (gold/mercury electrode) and coulometric (carbon electrode) methods are also used.

Gas-liquid chromatography-mass spectrometry (GC-MS)

Deuterated internal standard (D₈-homocysteine) is added to the plasma samples, homocysteine is released due to reduction with 1,4-dithio-D,L-threitol and the sample is purified by solid phase extraction using column chromatography with an anion exchange resin. Homocysteine is then derivatized with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide, and the butyldimethylsilyl derivatives are separated by means of capillary gas chromatography (using an automatic sample injection system) and identified and determined with a mass detector using the single ion monitoring (SIM) mode /3/.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The LC-MS/MS method is a novel method of homocysteine determination characterized by high specificity and sensitivity and good analytical precision. A daily throughput of up to 200 plasma samples can be handled and sample processing is less time consuming (40 samples in less than 1 h) /4/.

Capillary electrophoresis

This determination method uses laser-induced fluorescence detection. For sample processing, plasma samples are mixed with internal standard, reduced with tri-N-butylphosphine and then deproteinized (trichloroacetic acid) and derivatized (5-bromomethyl-fluorescein). Electrophoretic separation takes approximately 7 min. /5/.

Immunological methods

Fluorescence polarization immunoassay (FPIA). This method is based on the principle of reductive release of homocysteine, the conversion of homocysteine to S-adenosylhomocysteine (SAH) catalyzed by SAH hydrolase and subsequent immunological detection with an SAH antibody. Besides serving as fluorescence polarization immunoassay, this method can also be used as latex-enhanced nephelometric assay. Compared to the standard method HPLC, immunological assays also show good correlations and assay characteristics within a concentration range of 7 to 40 μmol/L and CVs of 3–5% /6/.

Enzymatic method

The enzymatic homocysteine determination methods are based on the reactions of the homocysteine/methionine cycle described in Section 13.1-1 – Introduction. The detection limit and specificity of the assays are increased by enzymatic recycling. As in the other methods, oxidized homocysteine is first of all reduced to homocysteine by tris-(2-carboxyethyl)-phosphine (TCEP).

In the homocysteine assay catalyzed by cystathionine-β-synthase (Fig. 13.2-1 – Principle of the homocysteine assay catalyzed by cystathionine-β-synthase), homocysteine and serine are condensed to form cystathionine. Cystathionine is then converted by cystathionine-β-lyase to form homocysteine and pyruvate. The reduction of NADH absorption at 340 nm allows the calculation of the amount of pyruvate present and converted to lactate. In a different enzymatic homocysteine assay, homocysteine is not measured directly, but in a cyclical enzymatic assay method the co-substrate conversion product is measured instead of the co-substrate itself. In a reaction with S-adenosylmethionine (SAM) catalyzed by homocysteine methyl transferase (HMTase), homocysteine is converted to SAH. SAH is then hydrolytically cleaved by SAH-hydrolase to form homocysteine and adenosine. The adenosine formed in this cyclic reaction is measured by converting the adenosine by means of adenosine desaminase to inosine and ammonia. Glutamate is formed by ammonia and ketoglutarate in an NADH-dependent reaction catalyzed by glutamate dehydrogenase (GLD) and its reduction of absorption is measured at 340 nm based on NAD formation. The change in absorption at 340 nm is correlated to the homocysteine concentration in the sample.

13.2.3 Specimen

EDTA fasting plasma taken from EDTA blood after 12 h fasting. After sampling, cool the EDTA blood on ice, centrifuge within 30 min. and separate the plasma.

13.2.3.1 Oral methionine load

In this provocation assay, a repeat measurement of homocysteine is performed after 4 and 6 hours following oral methionine administration (Tab. 13.1-1 –Homocysteine level in fasting plasma following oral methionine load):

• Collection of EDTA fasting blood for determining the homocysteine baseline concentration (store cool, separate plasma within 30 min.)
• Oral administration of L-methionine (0.1 g/kg of body weight) in 200 mL of fruit juice and a small, methionine-poor snack
• Collect EDTA blood again 4 and/or 6 hours following methionine administration to determine the post-methionine load concentration.

The homocysteine level primarily reflects the cystathionine-β-synthetase activity and/or availability of vitamin B₆ /7/. By contrast, the fasting homocysteine levels are not sensitive for vitamin B₆ deficiency. Within a fasting homocysteine range of 12–15 μmol/L, a high number of individuals yield pathological findings following methionine loading (above 38 μmol/L) /8/.

13.2.4 Reference interval

It does not make sense to specify a reference interval because a graded relation has been found between risk increase and/or the manifestation of cardiovascular and other degenerative disease and homocysteine concentrations already above 10 μmol/L (dose-response relation) /12/. An increase in homocysteine concentration by 1 μmol/L, the risk increases by 6–7% /13/. Risk intervals considering the differentiation by prophylactic and therapeutic aspects have been defined for practical use. Tab. 13.1-1 – Homocysteine level in fasting plasma following oral methionine load shows a concentration-dependent classification of hyperhomocysteinemias.

13.2.5 Clinical significance

If moderately elevated homocysteine concentration is detected (12–30 μmol/L), a second analysis is recommended after 4–6 weeks. The intraindividual variability of homocysteine is generally very low. Repeated measurements in healthy individuals after 6–18 months show very good conformity with the baseline values, with an individual, non-significant deviation of only 0.85–1.2 μmol/L /14/. Despite the low variability, repeated measurements in the decisive interval can improve diagnosis.

Moderate hyperhomocysteinemia (above 12 μmol/L) has been reported in about 10% of the general population, up to 40% of patients with vascular disease and about 50% of the elderly above 65 years of age. Synergistic interactions between homocysteine and additional risk factors (smoking, arterial hypertension, diabetes, hyperlipidemia) result in a proportional increase in total risk. Consequently, it is especially important to identify individuals at high risk of degenerative vascular disease.

13.2.5.1 Treatment goals and options

Prophylaxis

Supplementation remains a recommended option for prophylaxis. Doses within the sense of prophylactic measures are listed in Fig. 13.2-2 – Decision tree for the diagnosis and prophylaxis/therapy in hyperhomocysteinemia. Moreover, dietary measures such as conversion to a vitamin-rich diet are recommended.

Therapy

In patients with manifest vascular disease, stroke, patients with neurodegenerative disease or cognitive impairment, the goal should be to reach a plasma homocysteine level below 10 μmol/L /12/. Homocysteine levels above 12 μmol/L, vitamin deficiency as well as functional disorders of the kidneys and thyroid should be excluded as causes of elevated homocysteine. The findings should be interpreted due to consideration of the influencing factors. See

• Tab. 13.1-1 –Homocysteine level in fasting plasma following oral methionine load
• Tab. 13.1-2 – Causes of homocysteine changes
• Tab. 13.1-3 – Mutations associated with hyperhomocysteinemia.

For example, a change of drugs, changed dose or treatment of hypothyroidism can result in marked lowering of homocysteine.

Recommendations for vitamin supplementation

The extent to which the homocysteine concentration is lowered by vitamin supplementation depends on the baseline concentration. Daily administration of 0.2–5 mg of folic acid can achieve a reduction by 16–39% /16/. Additional administration of vitamin B₁₂ is to avoid relative folate deficiency (i.e., in support of folate utilization).

Therapy with folic acid alone should not be given (especially not in the long term), but only in combination with vitamin B₁₂. Vitamin B₆ has little influence on the fasting homocysteine concentration, but is an important cofactor in catabolic transsulfuration and should, therefore, also be taken into account in B vitamin supplementation.

If homocysteine concentration is moderately elevated, daily vitamin supplementation of 0.2–0.8 mg of folic acid, 3–30 μg of vitamin B₁₂ (at least 100 μg in older patients due to malabsorption) and 2–20 mg of vitamin B₆ should be started. If a reduction into the range below 10 μmol/L of homocysteine is achieved, the homocysteine concentration can be checked every 1–2 years.

Other causes of increased vitamin requirement

If the decrease in homocysteine level is not satisfactory, other causes must be searched for. For example, renal and thyroid dysfunctions can be present besides vitamin deficiency. It should be noted that a vitamin B₁₂ concentration within the lower reference range does not exclude intracellular B₁₂ deficiency /14/. Genetic mutations of enzymes involved in metabolism, for example the most widely known 5,10 methylene tetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, can also cause higher vitamin requirement.

For other possible causes, see

• Tab. 13.1-1 – Assessment of homocysteine concentration in fasting plasma and following oral methionine load
• Tab. 13.1-2 – Causes of homocysteine changes
• Tab. 13.1-3 – Mutations associated with hyperhomocysteinemia.

Supplementation in renal dysfunction and enzyme deficiency

Very high vitamin doses (possibly also daily administration of 3 g of betaine) can be required in manifest renal dysfunction (insufficiency, dialysis), but in many cases it is not possible to reach normal concentrations. Homocysteine levels above 30 μmol/L detected in patients with no renal dysfunction can be caused by congenital enzyme deficiency, the prevalence of which has been underestimated to date. If a primary therapy attempt with pharmacological doses (e.g., 5–15 mg of folic acid, 1 mg of vitamin B₁₂ and more than 20 mg of vitamin B₆) fails, the patient should be referred to a specialist.

13.2.6 Comments and problems

Specimen

Plasma is to be preferred to serum. Sampling tubes other than those with added EDTA (e.g., 2-deaza-adenosine as stabilizer) are used in some cases to extend the period until centrifugation without significant change in homocysteine concentration. It is advisable for better comparability to pursue the principle of immediate separation by centrifugation or short-term cooled storage. On the other hand, 2-deaza-adenosine is not suitable as stabilizer for immunological assays.

There have been commercial efforts to use stabilizers for avoiding immediate centrifugation of the blood. Some manufacturers use SAH hydrolase inhibitors that inhibit the release of homocysteine from SAH. However, an increase in SAH yields incorrect homocysteine concentrations in immunological methods based on SAH determination /18/. In citrate monovette or primavette samples, the plasma homocysteine did not change significantly at 4 °C after 24 h and remained unchanged in the primavette at room temperature, but the SAH increased continuously /18/.

Sample processing after blood collection

After blood collection, homocysteine is generated in the blood cells and continuously released into the plasma, causing a gradual increase in plasma homocysteine concentration (5–15% per hour at room temperature) /18/. An increase in homocysteine can occur in serum samples due to cellular release during coagulation. The blood sample (typically EDTA blood) should always be stored in a cool place immediately after collection and, within 30 min, be centrifuged at 2,000 g for 10 min in a cooling centrifuge.

Method of determination

As a rule, total homocysteine is measured in plasma after a reduction step as the total of bound and free homocysteine. Therefore, the result does not take quantitative changes of the fractions into account.

Enzymatic assay: enzymatic homocysteine assays show good correlation with the HPLC method and the immunological assay over a large concentration range /6/. The coefficients of variation (day to day) reported for human sample specimens are below 7% for the low and high concentration ranges. Compared to the HPLC or immunological methods, the absolute values are slightly higher (up to 7% on average). The precision of the enzymatic assays verified against standard reference material has not always been satisfactory /6/. Moderately severe lipemia, icteric samples or hemolysis can affect the results /6/. Enzymatic homocysteine measurement in the plasma of patients with renal disease or patients with liver cirrhosis can be affected by interferences resulting in higher measurement results. In enzymatic assays based on the conversion of cystathionine to homocysteine and pyruvate, interference can be caused by elevated cystathionine levels in renal patients. In enzymatic homocysteine assays based on the conversion of ammonia to ketoglutarate in the indicator reaction, falsely high homocysteine concentrations can be measured in plasma with elevated ammonia concentrations (liver cirrhosis).

Influence factors

Moderately severe lipemia, hyperbilirubinemia or hemolysis can affect the assay quality /6/. Besides elevated homocysteine, elevated SAH can be measured in the plasma of patients with renal insufficiency. However, this error is not significant due to the markedly elevated homocysteine levels in these patients.

Other body fluids

Although no systematic analyses on the homocysteine concentration in other body fluids are available, smaller-scale studies provide information on homocysteine concentrations in other body fluids. Tab. 13.2-1 – Homocysteine concentrations in other body fluids shows selected information in this context. The homocysteine level in cerebrospinal fluid (CSF), determined using LC-MS/MS or HPLC, is about hundredfold lower than in plasma. CSF homocysteine is negatively correlated with serum and CSF folate, but positively correlated with CSF SAH and age. CSF and serum homocysteine are also correlated. CSF homocysteine has been reported to be higher in patients with Alzheimer’s dementia than in patients without neurological disease.

In a study, homocysteine levels in amniotic fluid showed comparable levels than in plasma (Tab. 13.2-1 – Homocysteine concentrations in other body fluids) /10/. It is reported that homocysteine concentrations in amniotic fluid are higher in small for gestational age newborns than in mature newborns. The homocysteine concentration in amniotic fluid correlates with fetal gestational age, femur length and biparietal diameter.

Determinations of the homocysteine concentration in ejaculate within the scope of research on in vitro fertilization showed that the homocysteine levels in the ejaculate were similar in fertile and idiopathic sub fertile men, but lower in sub fertile men /11/.

Drugs

Drug-induced alterations of homocysteine levels are to be taken into account (Tab. 13.1-2 – Causes of homocysteine changes). For example, elevated homocysteine can be caused by therapy with drugs containing SAM. Moreover, drugs such as methotrexate, carbamazepine, phenytoin, NO, anticonvulsives or 6-azauridine triacetate can affect the patient’s homocysteine metabolism causing elevated homocysteine concentrations.

Stability

If not immediately centrifuged, the blood can be stored on ice or in the icebox for up to 1 h after collection. Without quick centrifugation and separation of the blood cells, the homocysteine increases rapidly as a function of temperature and time, resulting in falsely elevated measurement results. After centrifugation, homocysteine in plasma is stable: for 24 h at room temperature, for up to one week in the icebox (2–8 °C), for months at –20 °C, for up to 10 years at –70 °C /19/.

13.2.7 S-Adenosyl homocysteine (SAH), S-Adenosyl homocyteine (SAM)

SAH is formed via de methylation of SAM and is a potent inhibitor for many transmethylation reactions. SAM (activated methionine) plays an important role in intermediary metabolism as universal methyl group donor. It has been pointed out in recent publications that SAH might be a better biomarker for the development of Alzheimer’s dementia than homocysteine /20/. In a study on Alzheimer patients, SAH in CSF was reported to correlate significantly with phosphorylated tau protein as the key protein for the development of Alzheimer’s dementia, whereas no such association was found for homocysteine /21/. It was shown that biomarkers of neurodegeneration (amyloid precursor protein (APP) and α-synuclein) are correlated to biomarkers of methylation (SAH and SAM) in patients with Parkinson’s disease /22/. Better cognitive function was related to higher methylation potential (SAM/SAH ratio) /22/. It has been suggested that hypo methylation as an essential pathomechanism in hyperhomocysteinemia might be better reflected by SAH or the SAM/SAH ratio than by homocysteine.

The fast and reliable stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous quantification of SAM and SAH in body fluids /23/. The method comprises a phenylboronic acid-containing solid-phase extraction procedure, serving for binding and clean-up of SAM and SAH. After extraction, SAM and SAH are separated using UPLC (Acquity UPLC BEH C₁₈ column) and quantified using the tandem mass spectrometer.

The pre analytical procedures are to be followed precisely because SAM is unstable and degraded to form SAH. The EDTA blood collected for SAM and SAH determination should (as for homocysteine) be stored in a cool place until centrifugation, centrifuged within 30 min., and the plasma should be separated immediately and acetic acid be added for deproteination (1 mL of plasma + 0.1 mL of 1 N acetic acid). After thorough mixing, the mixture should be centrifuged again and the supernatant be stored at –70 °C until analysis. Plasma concentrations in normal individuals were 85.5 ± 11.1 nmol/L for SAM and 13.3 ± 5.0 nmol/L for SAH. The UPLC-MS/MS method is highly sensitive and specific and provides good precision and accuracy.

13.2.8 Pathophysiology

Based on different quantification approaches, up to 25% of cardiovascular events might be prevented by lowering elevated homocysteine concentrations /24/. Besides its significance as a risk factor with additional prognostic function, homocysteine is a sensitive diagnostic biomarker for folate, vitamin B₁₂ and vitamin B₆ deficiency /25/. Moreover, the determination of homocysteine is suited for documenting successful therapy following vitamin supplementation. In addition to their function as co factors for the enzymes involved in homocysteine metabolism, the vitamins B₁₂, B₆ and folic acid have important, independent properties. Folic acid and vitamin B₆ deficiency are independent risk factors for cardiovascular disease.

Low 5-methyl-THF

Low folate concentration, a mutation in the MTHFR gene or low vitamin B₁₂ concentration can cause impaired synthesis of 5-methyl-THF resulting in impaired remethylation of homocysteine to methionine /26/. It has been attempted for compensation to increasingly catabolize homocysteine using transsulfuration. However, the increased amount of formed homocysteine cannot be converted by transsulfuration because the SAM concentration is too low to activate the catabolic metabolic pathway. The increase in homocysteine is additionally enhanced by the low 5-methyl-THF concentration that does not inhibit the de methylation of SAM and thus contributes to further lowering of SAM.

Elevated 5-methyl-THF

Vitamin B₁₂ or methionine synthase deficiency inhibit the remethylation of homocysteine to methionine /27/. The resulting elevated 5-methyl-THF inhibits the de methylation of SAM, and elevated SAM concentrations activate cystathionine-β-synthetase. Hyperhomocysteinemia in vitamin B₁₂ deficiency is slightly less pronounced than in folate deficiency because the homocysteine catabolism in vitamin B₁₂ deficiency is more active than in folate deficiency.

Chronic renal insufficiency

Premature atherosclerotic vascular disease in renal patients are partly explained with the high prevalence of hyperhomocysteinemia. The median homocysteine level in patients with hemodialysis and peritoneal dialysis is approximately 30 μmol/L /28/. In many cases, kidney transplantees also have moderately elevated homocysteine (median: 20 μmol/L). Impaired remethylation of homocysteine to methionine (only partly reversible by vitamin supplementation) has been discussed as the main cause of the high prevalence of hyperhomocysteinemia in renal patients (about 70% of transplantees and about 90% of patients with hemodialysis) /29/. Besides renal dysfunction, renal patients also have substantial B vitamin deficiency (B₁₂, B₆, folate) /28/.

Moderately impaired transsulfuration

Transsulfuration is only moderately impaired in vitamin B₆ deficiency or in the presence of a heterozygous defect in the Cystathionine-β-synthetase gene /26/. In combination with non-impaired remethylation, the residual transsulfuration activity prevents hyperhomocysteinemia as long as the homocysteine load remains low. However, pronounced hyperhomocysteinemia develops following food intake or after a methionine load test with existing vitamin B₆ deficiency. Therefore, the methionine loading test is primarily used for identifying individuals with impaired transsulfuration, who have a normal or borderline homocysteine level under fasting conditions.

References

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9. Obeid R, Kostopoulos P, Knapp JP et al. Biomarkers of folate and vitamin B₁₂ are related in blood and cerebrospinal fluid. Clin Chem 2007; 53: 326–33.

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13. Bots ML, Launer LJ, Lindemans J et al. Homocysteine and short-term risk of myocardial infarction and stroke in the elderly: the Rotterdam Study. Arch Intern Med 1999; 159: 38–44.

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16. Homocysteine Lowering Trialists’ Collaboration. Lowering blood homocysteine with folic acid based supplements: meta-analysis of randomised trials. BMJ 1998; 316: 894–8.

17. Herrmann W, Schorr H, Bodis M et al. Role of homocysteine, cystathionine and methylmalonic acid measurement for diagnosis of vitamin deficiency in high-aged subjects. Eur J Clin Invest 2000; 30: 1083–9.

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13.3 Vitamin B₁₂, Holotranscobalamin

Wolfgang Herrmann, Rima Obeid

Vitamin B₁₂, also called cobalamin is a cofactor of two enzymes methionine synthase and L-methyl-malonyl-coenzyme A mutase. The basic structure of cobalamin forms is shown in Fig. 13.3-1 – Basic structure of cobalamin forms.

Methionine synthase forms methionine from homocysteine and 5-methyltetrahydrofolate.

L-methylmalonyl-CoA-mutase catalyzes the formation of methyl malonic acid from succinyl-CoA.

The main dietary sources of vitamin B₁₂ are liver, meat, fish, milk, cheese and eggs.

Vitamin B₁₂ is necessary /1/:

• Together with folate, for the maturation of erythropoietic cells. Deficiency in either of the two vitamins causes dyssynchrony between maturation of cytoplasm and that of nuclei and leads to macrocytosis, immature nuclei, and hyper segmentation in granulocytes.
• For the development and initial myelination of the central nervous system as well as for maintenance of its normal function. Vitamin B₁₂ deficiency can lead to demyelination of the cranial and thoracic dorsal and lateral columns of the spinal cord, occasional demyelination of cranial and peripheral nerves, and demyelination of white matter in the brain.

About 80% of serum vitamin B₁₂ are bound to the transport protein haptocorrin and are, in this form, metabolically inactive. The residual proportion is bound to trans cobalamin and biologically active (holotranscobalamin).

13.3.1 Indication

Populations at risk for vitamin B₁₂ deficiency are listed in Tab. 13.3-1 – Populations at risk for vitamin B₁₂ deficiency.

13.3.2 Method of determination

Besides total vitamin B_12, holotranscobalamin (holoTC), the active form of vitamin B₁₂ is clinically relevant for determination of the vitamin B₁₂ status.

13.3.2.1 Total vitamin B₁₂

Chemiluminescence immunoassay

Competitive immunoassay with direct chemiluminescence detection. Vitamin B₁₂ is released from its endogenous protein binding site by reduction in an alkaline environment. Free vitamin B₁₂ in the patient sample competes with a defined amount of labeled vitamin B₁₂, (e.g., labeled with acridiniumester, for binding sites of a limited amount of pure intrinsic factor). The intrinsic factor binds to paramagnetic particles as the solid phase. After magnetic separation, the chemiluminescence reaction is triggered. There is an inversely proportional relationship between the amount of vitamin B₁₂ in the patient sample and the measured relative light units.

Solid phase radioimmunoassay

Reaction tubes coated with antiserum against vitamin B₁₂/bovine serum albumin are used. A radio iodinated tyrosine methyl ester of vitamin B₁₂ (B₁₂-TME-¹²⁵I) is incorporated as radioactive tracer. In the competitive assay, endogenous vitamin B₁₂ and B₁₂-TME-¹²⁵I compete for the antibody binding sites on the vessel wall. The radioactivity measured after rinsing of the reaction tubes is reversely proportional to the vitamin B₁₂ concentration in the sample /2/.

Competitive radio ligand assay

Endogenous vitamin B₁₂ is extracted from the binding proteins in the serum and a defined amount of isotope-labeled vitamin B₁₂ and vitamin B₁₂-binding protein ( e.g., IF is added). Then, the free and the protein-bound vitamin B₁₂ fractions are separated. The radioactivity in the free fraction is reversely proportional to the vitamin B₁₂ concentration in the sample.

13.3.2.2 Holotranscobalamin (holoTC)

Holotranscobalamin= total vitamin B₁₂ = TVB12

Enzyme immunoassay I

This method is based on the micro particle enzyme immunoassay (MEIA) principle using an analyzer for measurement. The assay uses micro particles coated with monoclonal anti-holoTC antibodies /4/. HoloTC antigen in the sample binds to the coated micro particles forming an antigen-antibody complex on the micro particles. Alkaline phosphatase (ALP) conjugate is added and, after a washing step, the substrate 4-methylumbelliferyl-phosphate is added to the matrix cell. The ALP-labeled conjugate catalyzes the cleavage of a phosphate group from the substrate. The resulting fluorescent product, 4-methylumbelliferone, is measured by the optical assembly of the MEIA. The signal is directly proportional to the holoTC concentration in the sample /5/.

In a different method, magnetic beads coated with vitamin B₁₂ precipitate apo-transcolobalamin in the plasma. The holoTC present in the supernatant is measured by ELISA. ELISA uses immobilized anti-human TC (from rabbit) to capture and detect holoTC in the plasma with a biotinylated antibody in a horseradish peroxidase avidin-mediate color reaction. The color reaction is directly proportional to the plasma holoTC concentration /6/.

Radioimmunoassay

HoloTC is bound by a monoclonal anti-TC II antibody immobilized on magnetic micro spheres and then magnetically separated from the non-bound fraction remaining in the liquid phase. Cobalamin is released by reduction and extraction from the magnetically separated holoTC, a defined amount of ⁵⁷Co-tracer is added, and labeled cobalamin and unlabeled cobalamin compete for binding sites on immobilized intrinsic factor. After the non-bound fractions have been washed out, the radioactivity is measured. The radioactivity is inversely proportional to the holoTC concentration in the sample /7/.

13.3.2.3 Methyl malonic acid

Gas-liquid chromatography-mass spectrometry (GC-MS)

Deuterated internal standard is added to the samples, and the samples are purified by solid phase extraction using column chromatography with an anion exchange resin. MMA is then derivatized with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide and the derivatives are separated by means of capillary gas chromatography and identified and quantified with a mass detector using the single ion monitoring (SIM) mode /8, 9/. Besides MMA, homocysteine is also used as a B₁₂ marker. However, MMA is more specific because the homocysteine concentration increases in B₁₂ and folate deficiency.

13.3.2.4 Vitamin B₁₂ resorption test

Principle: the so-called Schilling test analyzes urinary excretion of orally administered, radioactively labeled vitamin B₁₂ /10/.

Preparation: any vitamin B₁₂ supplementation is discontinued at least 2 days before the test. If the patient received radioactive substances in the recent past before the test, 24-h urine is collected and checked for radioactivity before administration of the capsule with ⁵⁷Co-vitamin B₁₂.

Test procedure: the bladder is emptied and the fasted patient is given a ⁵⁷Co- or ⁵⁸Co-vitamin B₁₂ capsule with an activity of about 20 kBq. This is followed 2 hours later by intramuscular injection of 1 mg of vitamin B₁₂. Urine is collected for 24 h and the urine volume is measured. The total activity excreted in urine is correlated to the orally administered dose.

Clinical significance: the percentage excretion of labeled vitamin B₁₂, related to the administered dose, is evaluated. The Schilling test is used to clarify the causes of vitamin B₁₂ deficiency. In case of low radioactivity excretion, an extended test can be added for differentiation between malabsorption and intrinsic factor deficiency. The patient is administered the ⁵⁷Co-vitamin B₁₂ capsule and 35 mg of intrinsic factor concentrate at the same time.

13.3.2.5 Deoxyuridine suppression test

The deoxyuridine suppression test provides objective evidence of impaired DNA synthesis in patients with megaloblastic anemia and can be used for differentiation between vitamin B₁₂ deficiency and folate deficiency. The test analyzes the capacity of leukocytes/bone marrow cells for incorporation of ³[H]-thymidine into their DNA, both before and after adding the corresponding cofactor (vitamin B₁₂ or folate). The rate of incorporation is compared with that of control cells. Normoblastic bone marrow cells incorporate less than 10% of ³[H]-thymidine and megaloblastic bone marrow cells incorporate more than 10% of the controls /11/.

13.3.3 Specimen

Vitamin B₁₂, holoTC, MMA, homocysteine:

• For the separate determination of individual biomarkers: 1 mL of serum or plasma, respectively
• For the combined determination of holoTC, MMA and homocysteine: 2 mL of serum or plasma.

Schilling test: refer entire 24-hour urine sample without supplement to the laboratory.

Deoxyuridin suppression test: leukocytes or bone marrow cells.

13.3.4 Reference interval

Refer to

• Tab. 13.3-2 – Age- and gender-specific reference intervals for vitamin B₁₂
• Tab. 13.3-3 – Reference intervals for biomarkers of the vitamin B₁₂ status.

13.3.5 Clinical significance

The prevalence of vitamin B₁₂ deficiency is high in at-risk populations with sometimes irreversible consequences. In patients with low holo TC concentration (< 400 pmol/L) physicians are not sure whether there is a deficiency of vitamin B₁₂ and functional vitamin B₁₂ (methylmalonic acid, MMA) is ordered. Commercially available assays evaluate functional vitamin B₁₂ using the determination of MMA.

Laboratory evaluation on vitamin B₁₂ deficiency:

• The first step is to determine the vitamin B₁₂ serum level. Falsely positive and falsely negative results (up to 50%) can be of disadvantage if the reference interval of a given test is used for assessment.
• If vitamin B₁₂ deficiency is suspected clinically at levels within the reference interval, the determination of holotranscobalamin or methylmalonic acid is recommended.

Refer to Fig. 13.3-2 – Algorithm for diagnosis of vitamin B₁₂ deficiency.

13.3.5.1 Assessment of makers of vitamin B₁₂ deficiency

Determination of total vitamin B₁₂ is a first line marker, but provides limited diagnostic specificity and sensitivity, especially in individuals with vitamin B₁₂ levels below 400 pmol/L /12, 13/. A total vitamin B₁₂ level within the lower reference interval between 156–400 pmol/L suggests that vitamin B₁₂ deficiency cannot be excluded /5, 14/. It has been shown that individuals with vitamin B₁₂ levels within the reference range (above 156 pmol/L) may have symptoms of vitamin B₁₂ deficiency /16, 17/ and that individuals with normal vitamin B₁₂ concentrations can have elevated MMA concentrations (above 300 nmol/L) and low holoTC concentrations indicating intracellular, metabolically manifest (functional) vitamin B₁₂ deficiency /13, 18/. Conversely, normal MMA in low vitamin B₁₂ concentration as falsely positive finding is possible.

Isolated low serum holoTC concentration is considered to be the earliest marker of vitamin B₁₂ deficiency and indicates that the body has a low vitamin B₁₂ supply and vitamin B₁₂ stores are being depleted /13/. Consequently, low holoTC is indicative of a negative B₁₂ balance. Clinical or hematological manifestations are not yet found in this stage. In metabolically manifested vitamin B₁₂ deficiency [low holoTC, elevated MMA (and homocysteine)], there still may be no clinical symptoms. The diagnostic use of holoTC allows early treatment before irreversible neurological damage occurs.

HoloTC as metabolically active vitamin B₁₂ fraction correlates significantly with MMA. Vitamin B₁₂ , on the other hand, correlates significantly with holoTC within the reference interval, but less within the low concentration range /13/. Therefore, holoTC and MMA are better suited for the detection of vitamin B₁₂ deficiency than total vitamin B₁₂ /5, 14, 19/.

In a second study /19/ patients with a Holo TC level < 300 pmol/L were investigated for MMA. A positive correlation was found between Holo TC level and MMA in 71% of cases. A disagreement was evaluated between Holo TC level und MMA in 18.8% of cases. The optimized cut-off value for MMA was 45.5 pmol/L. The diagnostic sensitivity for MMA was 66.7%, the diagnostic specificity 60%, the positive predivtive value 30.7% ,and the negative predictive value 88.9%.

13.3.5.2 Stages and early detection of vitamin B₁₂ deficiency

The development of vitamin B₁₂ deficiency passes through various stages /20/. The stage of store depletion in the plasma and cells (negative vitamin B₁₂ balance) is followed by functional vitamin B₁₂ deficiency, in which metabolic disorders occur indicated by elevated homocysteine and/or MMA.

Store depletion

Store depletion is solely reflected by the holoTC level, which is low in this stage.

Functional B₁₂ deficiency

Because of depleted stores, the cell is no longer capable to have a balanced vitamin B₁₂-dependent metabolism. Besides low holoTC and holohaptocorrin, the metabolic biomarkers homocysteine and MMA are elevated, where MMA is a sensitive, but not specific biomarker of functional vitamin B₁₂ deficiency /12/. In addition, homocysteine is found to be elevated in folate or vitamin B₆ deficiency.

Clinical B₁₂ deficiency

The clinical manifestation stage is characterized by macrocytic anemia with elevated MCV, decreased MCHC and neutrophil hyper segmentation. Even persistent functional vitamin B₁₂ deficiency does not necessarily result in clinically abnormal hematological manifestation. Vitamin B₁₂ deficiency can cause neurological damage without the presence of classic hematological changes /20/. However, metabolic disorders primarily caused by vitamin B₁₂ deficiency, such as hyperhomocysteinemia and hypo methylation, are considered to be important factors in the development of neurodegenerative diseases /21/. Early diagnosis of vitamin B₁₂ deficiency is important not only because neurological damage is irreversible, but also because metabolic disorders can easily be corrected by adequate vitamin supplementation.

Early diagnosis is possible with modern biomarkers such as holoTC and MMA. However, a reliable diagnosis of vitamin B₁₂ deficiency cannot be achieved based on individual biomarkers. The combined use of the biomarkers holoTC, MMA and homocysteine provides diagnostic validity.

In a study on adults with serum vitamin B₁₂ ≤ 221 pmol/L, holoTC was determined in parallel /4/. 68% of the individuals in the lower normal range of vitamin B₁₂ (139–221 pmol/L) showed holoTC ≤ 40 pmol/L. Only 40% of the group with pathologically low total vitamin B₁₂ also had holoTC ≤ 40 pmol/L. In this study, a diagnostic sensitivity of 86% and a specificity of 66% were reported for holo TC (threshold 40 pmol/L).

False low holotranscobalamin levels in patients with normal vitamin B₁₂ concentration support the presence of a homozygous single point mutation c.855T>A in exon 6 of the TCN2 gene, corresponding to a asparagine to lysine substitution in position 267 of the mature protein /24/.

13.3.5.3 Causes of vitamin B₁₂ deficiency

As a coenzyme, vitamin B₁₂ is involved in metabolic processes in hematopoiesis, the development of the nervous system and regeneration of mucus layers. It is directly interrelated with folate metabolism because methyl cobalamin catalyzes the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine for methionine synthesis. Except in vegetarians, who have a life-style related vitamin B₁₂ deficiency, it is mostly caused by intestinal malabsorption or disturbed formation of intrinsic factor. The classic vitamin B₁₂ deficiency is pernicious anemia initially manifested as fatigue, jaundice and palpitation.

Using more sensitive and more specific biomarkers such as MMA or holoTC, it has been shown that the prevalence of subclinical functional vitamin B₁₂ deficiency in the population is higher than assumed to date /22, 23/. The prevalence of vitamin B₁₂ deficiency in younger adults is reported 5 to 7% /23/. Functional B₁₂ deficiency [elevated MMA and low holoTC (vitamin B₁₂)] is common in older individuals and was diagnosed in elderly, healthy individuals above 65 years of age with a prevalence of 10–30% /22, 23, 26/.

It has been reported that despite complying with the recommended dietary intake of vitamin B₁₂ (> 2.4 μg/day) in most cases, elderly adults show a high prevalence of slightly abnormal vitamin B₁₂ status (elevated MMA and homocysteine) attributed to non-dietary causes (such as malabsorption) /27/. It was shown in elderly patients from Strassburg with vitamin B₁₂ deficiency that 53% had vitamin B₁₂ malabsorption, 33% had pernicious anemia, only 2% had diet related B₁₂ deficiency and 11% had B₁₂ deficiency of unclarified etiology /28/. It should be noted that, because of the low recommended dietary allowance B₁₂ dietary insufficiency in older people is underestimated.

Children of mothers with vitamin B₁₂ deficiency can be born with a deficiency or, if fed exclusively with breast milk, can acquire the deficiency within the first 4–6 months. Typical symptoms of vitamin B₁₂ deficiency are deficits in brain development, growth, general development, hypotension, lethargy, tremor, hyperexcitability and anemia. Evidence of reduced myelination and atrophy has been provided by brain imaging. Vitamin B₁₂ substitution achieves rapid improvement /1/. However, permanent impairment is increasingly likely with longer lasting vitamin deficiency.

In vegetarians, the prevalence of abnormal vitamin B₁₂, MMA or holoTC depends on the duration and strictness of the vegetarian diet /13, 32/. In a study on lacto vegetarians and lacto-ovo vegetarians, 63% of the individuals showed elevated MMA (above 271 nmol/L), 73% a holoTC concentration ≤ 35 pmol/L and 33% a homocysteine concentration above 12 μmol/L. In vegans, elevated MMA was detected in 86%, low holoTC in 90% and hyperhomocysteinemia in 55% of the individuals /13/.

13.3.5.4 Differential diagnosis of vitamin B₁₂ deficiency

In order to clarify the causes of vitamin B₁₂ deficiency confirmed by laboratory and clinical findings, further laboratory tests should be performed.

• Antibodies against parietal cells: based on determination using ELISA in pernicious anemia, these antibodies have a diagnostic sensitivity of 80% at 50% specificity decreasing with age.
• Antibodies against IF: in pernicious anemia, these antibodies have a diagnostic sensitivity of 40% at 90% specificity
• Determination of gastrin: the determination of gastrin can help in many cases, if negative parietal and intrinsic factor antibody results are negative. High concentrations of gastrin indicate achlorhydria in pernicious anemia
• Vitamin B₁₂ resorption test (Schilling test): the test is helpful for the detection and differentiation of B₁₂ malabsorption. Reduced urinary vitamin B₁₂ excretion following oral administration of radioactive vitamin B₁₂ is usually based on intrinsic factor deficiency caused by autoimmune destruction of the parietal cell function in pernicious anemia. Intrinsic factor deficiency is confirmed by normalization of the assay upon concurrent administration of intrinsic factor and radioactive vitamin B₁₂. If the test still yields pathologic results, it can be repeated following treatment with antibiotics or vermicides to eliminate, for example, intestinal bacterial overgrowth or infestation with fish tapeworm. Continuously pathologic results are evidence of disease of terminal ileum (e.g., Crohn’s disease).

Diseases and defects associated with vitamin B₁₂ deficiency are shown in Tab. 13.3-4 – Diseases and defects associated with vitamin B₁₂ deficiency.

13.3.6 Comments and problems

Sample material

Serum can be stored in the dark for 1 day at 2–8 °C (longer storage at –20 °C). Hemolytic samples affect the test result and should not be used; lipemic samples are to be centrifuged at 13,000 × g prior to the test.

Reference interval

Because the reference intervals for the assays are different, the intervals specified for a given assay and manufacturer should be noted. The reference intervals for the assay published in the literature are only to a limited extent applicable to immunoassay findings. The reference intervals for adults shown in Tab. 13.3-2 and Tab. 13.3-3 refer to chemiluminescence immunoassay and those for children refer to radioimmunoassay.

The reference interval of the microbiological method differs from the information shown in Tab. 13.3-2 [adults 25–500 ng/L (18.4–500 pmol/L), immunoassay] /2/.

Diagnostic efficiency of vitamin B₁₂, holoTC, MMA

The diagnostic sensitivity and specificity of vitamin B₁₂ assays are not satisfactory. Values in the lower reference interval, in particular, are not conclusive /13, 35/. For example, about 25% of patients with vitamin B₁₂ below 148 pmol/L (200 ng/L) show no clinical symptoms of vitamin B₁₂ deficiency /36/. If MMA is used as biomarker of functional B₁₂ deficiency, 90–95% of the patients with elevated MMA also have low vitamin B₁₂ /34/.

In studies on vegetarians, the diagnostic efficiency of detecting vitamin B₁₂ deficiency was about 40% higher when using MMA instead of vitamin B₁₂ assay /13/. By contrast, diagnostic comparison yielded close agreement between MMA and holoTC.

In assays on serum samples with unconfirmed vitamin B₁₂ deficiency, samples with normal creatinine tested for elevated MMA (≥ 300 nmol/L) showed a significantly larger area under the receiver operating characteristic (ROC) curve compared to total vitamin B₁₂ (cutoff ≤ 156 pmol/L), 0.71 vs. 0.60 /14/. Accordingly, the diagnostic sensitivity and specificity were higher for holoTC than for total vitamin B₁₂. In studies on younger, healthy individuals [median age 34 (21–68) years], low vitamin B₁₂ levels were detected in 1% of the individuals, whereas holoTC was low in 11% and MMA was elevated in 5% /12/. Consequently, the prevalence of functional vitamin B₁₂ deficiency was significantly higher than that of low vitamin B₁₂ levels.

Vitamin B₁₂ deficiency has been reported to increase significantly with age /22/. In elderly individuals [median age 82 (69–92) years], low vitamin B₁₂ levels were found in 7%, but low holoTC concentrations in 21% and high MMA concentrations in 42% of the subjects /25/. Besides the elderly, vegetarians and patients with renal disease are particularly affected by a high prevalence of vitamin B₁₂ deficiency /13/.

Individuals with increased vitamin requirement are another group at risk of B₁₂ deficiency, for example pregnant and lactating women, patients with autoimmune disease or with HIV infection.

Moreover, B₁₂ deficiency can also develop under intake of proton pump inhibitors /13/.

Examinations of gastrectomized patients yielded holoTC below 42 pmol/L in 25% compared to total vitamin B₁₂ below 139 pmol/L in only 7.8% of the individuals /15/. Moreover, it was found that 44% of these patients with borderline total vitamin B₁₂ (139–295 pmol/L) had low holoTC (below 42 pmol/L) /15/.

Limitations in renal patients

Discrepant findings between the metabolic biomarker MMA and holoTC and vitamin B₁₂ have been reported in patients with chronic kidney disease. Markedly elevated MMA were found, while serum levels of holoTC or vitamin B₁₂ were elevated /25, 30/. Refer to Fig. 13.3-3– Decrease in methylmalonic acid in patients with kidney disease after injection of vitamin B₁₂.

The folate form distribution in women (pregnant women and control cohort), umbilical cord blood and older adults is shown in Tab. 13.4-5 – Folate forms in serum using UPLC-MS/MS.

Similar constellations have also been observed in older individuals with impaired renal function. In this case, increased concentrations of circulating vitamin B₁₂ are necessary to meet the intracellular requirement of vitamin B₁₂ /25, 30/. Consequently, the reference intervals for holoTC and vitamin B₁₂ do not apply to renal patients. Moreover, the utilization of MMA as a metabolic biomarker of vitamin B₁₂ in renal patients is only possible to a limited degree. In renal patients, the MMA concentration decreases as a function of increasing circulating vitamin B₁₂ concentration until a vitamin B₁₂ resistant level is reached. Beyond this level, further increase in vitamin B₁₂ in plasma will not achieve a further reduction in MMA concentration /30, 38/. However, elevated MMA can also have other causes, such as intestinal bacterial overgrowth.

13.3.7 Pathophysiology

The term vitamin B₁₂ (cobalamin, Cbl) summarizes porphyrin-like compounds consisting of four reduced pyrrole rings with a central cobalt atom (Fig. 13.3-1 – Basic structure of cobalamin forms). The corrin ring carries an α-axial and a β-axial ligand at the cobalt atom. Only cobalamines with an α-axial ligand consisting of 5,6-dimethylbenzimidazole have a vitamin function in the human body /37/. In medical literature, the term vitamin B₁₂ summarizes all cobalamines with a biological effect in the human body.

Corrinoids are usually classified as primitive coenzymes. Substitution at the β ligand of the cobalt atom by cyanide creates cyanocobalamin (CNCbl), substitution by OH¯ creates hydroxo-Cbl, substitution by CH₃ creates methyl-Cbl and substitution by 5-deoxyadenosyl creates 5’-deoxyadenosyl-Cbl (Fig. 13.3-1 – Basic structure of cobalamin forms). The final molecules of human biosynthesis are 5’-deoxyadenosyl-Cbl and methyl-Cbl₁₂. In humans, only two enzymes are catalyzed by vitamin B₁₂ -methionine synthase requiring methyl-Cbl as a cofactor, and L-methyl malonyl-CoA mutase, requiring adenosyl-Cbl as a cofactor. Methionine synthase catalyzes the re methylation of homocysteine to methionine, while L-methyl malonyl-CoA mutase catalyzes the isomerization of methyl malonyl-CoA to succinyl-CoA.

Various proteins are required for the transport and cellular uptake of vitamin B₁₂: intrinsic factor, trans cobalamin (TC), haptocorrin (also known as R-binder protein) and the membrane-bound intrinsic factor and TC receptors (Fig. 13.3-4 – Transport and cellular uptake of vitamin B₁₂).

In the stomach, vitamin B₁₂ is released from food proteins by the action of gastric acid and mainly bound to haptocorrin. In the upper intestine, the haptocorrin-cobalamin complex is degraded by the action of pancreatic enzymes and alkaline pH environment and vitamin B₁₂ is bound to a different protein, intrinsic factor (IF). In the lower ileum, the Cbl-IF complex is taken up into the cells via a special receptor on the surface of the enterocyte membrane. Vitamin B₁₂ absorption via this receptor is limited to below 3 μg per meal. About 1% of the food vitamin B₁₂ is absorbed independently of a receptor through passive diffusion.

Holotranscobalamine

In the enterocytes, the Cbl-IF complex is degraded and vitamin B₁₂ is transferred to a third protein, TC. This complex, referred to as holoTC, enters the bloodstream via portal circulation. Cellular internalization is effected via the TC receptor present on all cells. After internalization, holoTC undergoes lysosomal hydrolyzation, releasing cobalamin, which in the form of methyl-Cbl or adenosyl-Cbl activates the corresponding enzymes intracellularly by catalyzation. Cbl is released to the cytosol as Cbl³⁺ and there reduced to Cbl²⁺. Cbl²⁺ binds to apomethionine synthase and catalyzes the activity of this enzyme.

HoloTC is metabolically active vitamin B₁₂ with a relatively short biological half-life of about 1–2 hours. Only 10–30% of the vitamin B₁₂ circulating in the plasma are bound to TC (Fig. 13.3-5 – Cellular uptake and biological activity of vitamin B₁₂).

Approximately 80–90% of the plasma vitamin B₁₂ are bound to haptocorrin, which is also known as TC I and has a biological half-life of 9–10 days. This fraction of little metabolic activity does not contribute to the supply of peripheral cells with vitamin B₁₂ but is thought to be involved in transporting peripherally excessive vitamin B₁₂ back to the liver. TC III, an R-binder protein of the granulocytes, is a small fraction with a similar metabolic function as TC I.

Moreover, MMA as a sensitive biomarker of intracellular, functional vitamin B₁₂ deficiency plays a prominent role /35, 40/. In intracellular vitamin B₁₂ deficiency, homocysteine increases because of the impaired methionine synthase reaction and MMA increases because of the impaired L-methyl malonyl-CoA mutase catalyzed reaction (Fig. 13.3-6 – Cobalamin-dependent biological reactions in the cytosol and mitochondria and in cobalamin deficiency). Homocysteine also increases in folate and vitamin B₆ deficiency, while MMA is considered a specific and sensitive biomarker of functional vitamin B₁₂ deficiency /40/.

Clinical effects of vitamin B₁₂ deficiency

Because the human body has significant vitamin B₁₂ stores, it takes years before under supply becomes clinically evident. Vitamin B₁₂ deficiency generally passes through various stages (Fig. 13.3-7 – Cellular uptake, intracellular distribution and synthesis of cobalamin coenzymes). Besides its significance as a risk factor for cardiovascular and neurodegenerative disease, hyperhomocysteinemia in vitamin B₁₂ deficiency indicates vitamin deficiency and hypo methylation, for example, of DNA, RNA, myelin, phospholipids or neurotransmitters. Hypo methylation is caused by reduced availability of S-adenosylmethionine (based on the vitamin B₁₂ deficiency-induced inhibition of methionine synthase and reduced methionine) functioning as methyl donor for all methyltransferases. Hypo methylation of the basic myelin protein at position 107 (arginine), for example, can destabilize the protein and, thus, lead to the development of neuropathies. Funicular spinal disease is a common secondary neurological disease of Cbl deficiency. Psychiatric and neurological diseases, such as cognitive impairment, depression and dementia, can precede hematological anomalies by years or are even not detected.

Nevertheless, morphological changes in blood and bone marrow cells belong to the most important symptoms of vitamin B₁₂ deficiency. Because of the high cell turnover rate, hematopoiesis quickly and sensitively responds to blocked nucleic acid metabolism. Megaloblastic anemia due to vitamin B₁₂ deficiency develops based on defective DNA synthesis and resulting impaired nucleus maturing, while the cytoplasm develops normally. Cells with more or less strong morphological changes are detectable in the periphery (MCV above 110 fL, MCH above 40 pg). Studies have also shown that deficiency of vitamin B₁₂ (low total vitamin B₁₂ or holoTC) in the elderly is associated with significant atrophy of the brain /42/. Supplementation with B vitamins for two years in older adults with mild cognitive impairment clearly slowed the rate of brain atrophy. A higher rate of accelerated brain atrophy was associated with poorer cognitive performance /43, 44/.

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13.4 Folate (Vitamin B₉)

Wolfgang Herrmann, Susanne H. Kirsch, Rima Obeid

Folate is the general term for the water-soluble vitamin B₉ naturally found in foods such as leafy green vegetables, legumes, egg yolks, liver and some citrus fruits. This vitamin is essential for normal cell growth and replication /1/. The bio availability of naturally occurring folate is less than that of folic acid, a synthetic compound that is used as supplements and in fortified foods. The term folate, as used in this publication, encompasses folic acid, natural food folate and folate derivatives (Fig. 13.4-1 – Structure of folate derivatives).

13.4.1 Indication

Serum or erythrocyte folate should be determined in:

• Anemia, especially megaloblastic anemia
• Older adults
• Folate malabsorption
• Inflammatory bowel disease
• Chronic alcoholism
• Chronic liver disease
• Complications during pregnancy
• Previous complications during pregnancy (preeclampsia or HELLP syndrome)
• Atherosclerotic vascular disease
• Dementia, depression and cognitive impairment
• Hyperhomocysteinemia

13.4.2 Method of determination

Commercially available ligand assays are used to detect metabolically active folates. Folate is determined in serum or – in specific problems – in the erythrocytes. Determination is predominantly based on ligand assays. Some assays allow the simultaneous determination of folate and vitamin B₁₂. It is recommended to use liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the quantification of individual folate forms, such as 5-methyl-THF in serum and erythrocytes.

The whole-blood folate concentration is determined by hemolyzing 1 part of EDTA-blood with 20 parts of 1% ascorbic acid solution and measuring the folate concentration in the hemolysate. The resulting value is to be multiplied with 21 and divided by the hematocrit value. For determining the erythrocyte folate concentration, the serum folate concentration is subtracted from the whole-blood folate and related to the hematocrit value of the sample:

Whole-blood folate – serum folate × [(100 – hematocrit)/hematocrit]

In simultaneous determination of folate and vitamin B₁₂, the sample must be divided because ascorbic acid interferes with the determination of vitamin B₁₂.

Competitive immunoassay

Principle: a release reagent (e.g., DTT) is added to the patient sample to separate the folates from their endogenous binding proteins. The free folates of the sample then compete with added folic acid labeled, for example, with biotin or acridinium ester, for a limited amount of folic acid-binding protein, labeled, for example, with ruthenium or biotin. The folic acid-binding protein loaded with folic acid/folate is separated and measured.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The LC-MS/MS methods allow the quantification of folic acid and reduced folates in the mono glutamate or poly glutamate form and folate degradation products, such as p-aminobenzoic acid, from small amounts of patient specimens /2/. An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method /3/ allows the simultaneous quantification of folic acid, 5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF and THF from serum or whole-blood hemolysates.

Principle: for determining the whole-blood folate concentration, 1 part of EDTA blood is hemolyzed with 10 parts of a 10 g/L ascorbic acid solution and 0.2% triton X-100 and incubated for conversion of the polyglutamates to monoglutamates by means of endogenous conjugases in blood. The erythrocyte folate is also calculated according to the formula above.

Serum and blood hemolysate samples are mixed with commercially available isotope-labeled (¹³C₅) internal standards and processed by solid phase extraction (OASIS MAX). The sample is concentrated for better quantification of the minor folate forms. The concentration is determined in the acidic mobile phase using liquid chromatography, followed by detection in the mass spectrometer using positive electrospray ionization in the multiple reaction mode (MRM). Sample processing is fast and the duration of analysis per sample is 2.5 min.

13.4.3 Specimen

• Serum: 1 mL
• EDTA blood for folate determination in whole-blood hemolysate: 2 mL

For LC-MS/MS methods, 250 μL of serum or 200 μL of EDTA blood are required.

13.4.4 Reference interval

The serum folate concentration depends on the nutritional state. Consequently, reference intervals can only to a limited extent be applied to specific patient groups. Therefore, the values listed in Tab. 13.4-1 – Folate reference intervals for fasting serum are only guidance values, which are influenced by a given assay and by the folate status of the reference cohort.

Different reference ranges have been introduced for Europe and the United States because of the different eating habits (fortification of food with folic acid and widespread vitamin intake in the United States) (Tab. 13.4-2 – Serum and whole-blood folate reference intervals in the USA and in Europe).

13.4.5 Clinical significance

Folic acid fortification of stable food in the USA is seen to be the reason for different normal intervals of folate in US and Europe. Accordingly, folate deficiency cannot be excluded if folate concentrations are within the lower fraction of the reference interval.

13.4.5.1 Folate deficiency

Folate deficiency is present when the serum folate level is below 3.5 μg/L and below 250 μg/L in the erythrocytes /5/. However, the observed symptoms are mainly restricted to the stages of pre latent and latent folate deficiency, while the diagnosis of clinically manifest megaloblastic anemia is an exception (Tab. 13.4-3 – Stages of folate deficiency) /5/.

The determination of erythrocyte folate provides information on the long-term folate status /6/. Because erythrocytic folate is less dependent on short-term nutritional effects, non-fasting samples can also be analyzed. However, the analysis of erythrocyte folate has a lower precision.

Folate deficiency at first leads to reduced urinary folate excretion, followed by a decrease in serum concentration and, after 3–4 weeks, by a decrease in erythrocyte folate concentration. Hyper segmentation of the neutrophil granulocytes is observed after 10–12 weeks, months later followed by thrombocytopenia, leukopenia and finally macrocytic anemia.

Megaloblastic anemia caused by folate deficiency cannot be distinguished from vitamin B₁₂ deficiency in the blood count. Therefore, the presence of folate deficiency requires further diagnostic clarification as to whether vitamin B₁₂ deficiency is also present. Other effects of folate deficiency: changes in the mucous layer of the oral cavity, diarrhea, impaired growth, reduced immune response, impaired fertility, congenital malformations, such as neural tube defect, and neurological and psychiatric disorders.

Because of its strong dependence on food quality, the folate status should be determined in two tests at intervals of several days. A level above 5.5 μg/L (12.5 nmol/L) most likely indicates adequate folate supply. Folate requirement is higher in individuals homozygous for 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Normal plasma homocysteine concentrations are found when the serum folate is above 15 nmol/L and the vitamin B₁₂ status is adequate.

There are about 12–28 mg of folate in the human body, with approximately half being localized in the liver. About 10–90 μg of folate are excreted daily with the bile and practically completely reabsorbed. However, reabsorption is restricted in inflammatory bowel disease. The body’s reserves of folate are small (biological half-life ~100 days). In dietary folate withdrawal, the reserves are sufficient for about 3–4 weeks before the serum folate decreases and, after another 10–12 weeks, hyper segmentation of the neutrophil granulocytes is observed. Insufficient dietary intake and anti folate medication are the main causes of folate deficiency.

Folate deficiency is very common in Europe and promoted by lack of fresh fruit and vegetables. Good food sources for folate are, for example, green vegetables, cereal products, fruit, yeast and liver. Up to 90% of the folates can be lost in the processing of cereal products and other food /7/. Folates are also lost because folate is sensitive to heat, storage and light.

The recommendations of several specialist associations to eat five portions of fruit and vegetables a day (600–700 g) are hardly realizable /8/. The daily intake of folate from foods in Central Europe is currently between 230 and 280 μg (Fig. 13.4-2 – Folate intake in the population of various European countries) /9/; accordingly, a substantial part of the normal population does not reach the required amount of folate through their natural diet alone /10/. The recommended daily allowance (RDA) of folate is 400 μg (600 μg for pregnant women) for dietary folate equivalents (DFE).

Because of the under supply, functional disorders are to be expected, such as hyperhomocysteinemia, morphological cell changes (hyper segmentation of neutrophil granulocytes), neural tube defects, neurological symptoms and anemia. A mean daily intake of about 400 μg of folate equivalents would optimize all folate-dependent metabolism and maintain a low homocysteine concentration.

The mean homocysteine concentration reaches a steady-state level of almost 11.0 μmol/L at a folate intake of 400 μg/d /11/ or a serum folate concentration of about 14 nmol/L /12/.

In illness, folate deficiency can develop from inadequate intake, losses, malabsorption or increased requirement. Risk groups and diseases associated with folic acid deficiency are shown in Tab. 13.4-4 – Risk groups and diseases associated with folate deficiency.

13.4.6 Comments and problems

Sample material

Serum is recommended for analysis. Because serum folate levels are strongly dependent on the last food intake, serum from fasting blood should be used for determination. The blood samples should be sufficiently coagulated before centrifugation. Concentrations measured in heparin anticoagulated blood are higher than in serum. EDTA plasma is not recommended because it turned out to be unstable. For determining folate forms in serum or EDTA whole blood, the samples should be stored at –80 °C as quickly as possible after collection. Repeated freezing and defrosting of the samples should be avoided. Folates are sensitive to temperature. Therefore, samples should not be handled and stored at room temperature for an extended period of time.

Reference interval

The reference intervals for erythrocyte folate are shown in Tab. 13.4-2. The folate form distribution in women (pregnant women and control cohort), umbilical cord blood and older adults is shown in Tab. 13.4-5 – Folate forms in serum using UPLC-MS/MS.

Evidence has been provided using the LC-MS/MS method that the distribution of folate forms in the erythrocytes differs in the different MTHFR genotypes. Compared to 677 CC and CT genotypes, carriers of 677 TT polymorphism accumulate up to 43% non methyl THF in the total folate concentration (Tab. 13.4-6 – Folate forms in serum using UPLC-MS/MS and immunological method by MTHFR C677T genotype) /30/. Like erythrocyte folate, folate concentrations and folate form distribution in serum are dependent on the MTHFR genotype /3/, which may affect DNA methylation. The differences between serum and erythrocyte folates only become evident in a low folate status /30/.

Reference intervals for the determination of folate forms using LC-MS/MS have not been specified to date because of the differences among the measuring methods. Some guidance values for serum and erythrocyte folate are shown in Tab. 13.4-7 – Categories for 5-methyl -THF in fasting serum/plasma and erythrocytes using UPLC-MS/MS or LC-MS/MS. The folate in serum/plasma and the cerebrospinal fluid (CSF) is shown in Tab. 13.4-8 – Folate in serum/plasma and CSF using LC-MS/MS or immunological method.

Hemolysis

As hemolysis leads to falsely high values because of the high folate concentration in the erythrocytes, hemolytic serum is not suited for analysis.

Exposure to light

Folic acid and reduced folates are sensitive to light. 24 h exposure to light at room temperature decreases folate concentration by 12% if stored in a plastic tube and by 19% if stored in a separator tube /37/.

Stability

At 2–8 °C, the sample should not be stored for more than 2 days. Deep-frozen serum samples keep for 1 month at –20 °C and for longer at –80 °C. Folic acid is relatively stable, while reduced folates, especially THF and dihydrofolate, are unstable. They are immediately subject to inter conversion and degradation that are dependent on enzymes and/or influenced by oxygen presence, temperature, exposure to light and pH value. Care should always be taken, especially in the determination of folate form distribution in serum and erythrocytes, to be aware of changes in the folate profile resulting from sample processing and analysis. Therefore, the folate forms are in many cases subdivided into two groups, 5-methyl-THF and non-methyl-THF.

13.4.7 Pathophysiology

Folate

Folate plays an essential role in cell growth and proliferation. It provides methyl groups to homocysteine for conversion to methionine. It is also essential for the synthesis of purine and pyrimidine nucleotides. For this reason, folates play a key role in numerous cellular functions. Folate deficiency impairs DNA synthesis, causes hypo methylation and this results in the development of clinical conditions, such as megaloblastic anemia, neural tube defects and neurological damage.

Folic acid is the synthetic form of the vitamin. Chemically, it is pteroylmonoglutamic acid and contains pteroic acid as a substructure, which consists of pteridine and p-aminobenzoic acid. Depending on the number of glutamyl residues, pteroylmonoglutamate, -triglutamate, -heptaglutamate and/or -polyglutamate are distinguished. The pteridine ring can be present in the oxidized, di hydrated and tetra hydrated form.

Natural folates differ by:

• The hydration state of the pteridine ring
• Substitutions at N-5 and N-10 of the pteridine ring; various C₁ units, such as methyl, formaldehyde or formate groups can be bound
• The length of the glutamyl chain.

The parent substance of the folate coenzymes is 5,6,7,8-tetrahydrofolate (THF), which acts as universal C₁ acceptor. 5-Methyl-THF, 5,10-methylene-THF, 5,10-methenyl-THF, 5-formyl-THF, 10-formyl-THF and THF are the most important folate forms in the body. Unmetabolized folic acid can be detected in the blood after intake of more than 200 μg/d of folic acid; however, the significance of the presence of unmetabolized folic acid in serum has been controversially discussed. 5-Methyl THF occurs in serum predominantly as mono glutamate and intracellularly as poly glutamate-THF with or without C₁ units.

Uptake and metabolism

Folic acid is the most stable form of the vitamin with the highest oxidation stage and is quantitatively resorbed (more than 90%) in this form. Folates occur in all foods of plant and animal origin. For example, liver, vegetables (lettuce, spinach, asparagus, tomatoes, cucumbers) or cereals are rich in this vitamin. Food mainly contains polyglutamylfolates that are hydrolyzed to mono glutamate by a γ-glutamyl-carboxypeptidase (EC 3.4.19.9) in the brush border of the mucosa cells in the duodenum and upper jejunum for resorption. Resorption is optimal at pH 6. Transport across the mucosal membrane is mostly active; only 20–30% of the folates are taken up by passive diffusion. Via portal circulation, the folates pass to the liver where they are converted to the methylated form. The predominant form in the blood is 5-methyl-THF (as well as THF and 10-formyl-THF), which is transported bound to albumin, α₂-macroglobulin and transferrin.

The uptake of 5-methyl-THF into the erythrocytes is based on the principles of saturation kinetics using a membrane-bound carrier. Passage across the blood-brain barrier is similar; the folate concentrations in the cerebrospinal fluid being approximately equal /38/ to 3-fold higher than in serum (Tab. 13.4-8 – Folate in serum/plasma and cerebrospinal fluid). In the erythrocytes, the folates are present as poly glutamate, mostly with 4–7 glutamate residues that have a high affinity to deoxyhemoglobin. Folate concentrations in erythrocytes are about 20–40-fold higher than in serum. For retention, intracellular 5-methyl-THF is de methylated in a vitamin B₁₂-dependent reaction and then converted to poly glutamate.

Folates and C₁ metabolism

Folic acid itself is not biologically active, but THF and its derivatives are. They are the decisive coenzymes acting as acceptors and carriers of hydroxy methyl groups (activated formaldehyde) and formyl groups (activated formic acid). The C₁ residues coming from various metabolic processes are bound to THF and passed on to suitable acceptors for the synthesis of substances. THF-C₁ compounds are present in various oxidation stages and can be inter converted as shown in Fig. 13.4-3 – Function of folate forms in human cells.

Methylation reactions play a prominent role in metabolism utilizing the following donors: 5,10-methylene-THF provides the methyl group for the formation of thymidylate from d-uridylate during DNA synthesis, 5-methyl-THF provides the methyl groups for the methylation of homocysteine to methionine. In the liver, homocysteine can also be methylated by betaine. The methyl groups of betaine are provided by S-adenosylmethionine (i.e., indirectly by 5-methyl-THF).

All other methylation reactions are based on S-adenosylmethionine, the universal methyl group donor for various biosyntheses, such as:

• The formation of phosphatidylcholine, choline, carnitine, acetylcholine, adrenalin, creatine, thymine (methyluracil)
• The methylation of myelin, DNA, receptors and neurotransmitters.

Folate deficiency is attributed to

• Under supply due to inadequate intake
• Reduced folate resorption in the intestinal tract
• Increased consumption
• In existing vitamin B₁₂ deficiency (secondary folate deficiency).

Refer additionally to Tab. 13.4-9 – Causes of folate deficiency.

Cell dysfunction

Persistent folate deficit causes the depletion of the folate stores in serum and in the erythrocytes and leads to hyper segmentation of the neutrophil granulocytes, later followed by thrombocytopenia, leukopenia and finally, after months, macrocytic anemia. Impaired erythropoiesis is based on impaired nucleic acid synthesis, impaired maturation and, to a lesser extent, reduced hemoglobin synthesis.

During DNA synthesis, deoxyuridine mono phosphate (dUMP) is methylated to deoxythymidine mono phosphate (dTMP = thymidylate) by thymidylate synthase, with the methyl group provided by 5,10-methylene-THF. dTMP is successively converted to thymidine triphosphate (dTTP) and incorporated into the DNA. The folate deficiency-induced inhibition of thymidylate synthase causes reduced formation of dTMP. Increased incorporation of uracil into the DNA due to dTMP deficiency is corrected by repair mechanisms (splicing) (Fig. 13.4-4 – Effects of folate deficiency and/or vitamin B₁₂ deficiency on erythropoiesis). This can lead to chromosomal breakage. Moreover, DNA hypo methylation can cause the activation of tumor suppressor genes /41/. Tissues with fast proliferating cells (lymphatic tissue, intestinal brush border membrane, hair follicles or tumor tissue) constantly require dTMP.

Like dUMP, the 5-fluorodeoxyuridylate (FdUMP) used in cancer therapy binds to thymidylate synthase, but – unlike dUMP – is not released and therefore causes irreversible inhibition of the enzyme. dTMP deficiency leads to the death of fast proliferating cells.

Hematopoietic cells with folate or cobalamin deficiency undergo changes in morphology, protein synthesis and cell kinetics. The changes are especially pronounced in the final stage of cell division and in non-divisible cells and mainly affect erythropoiesis and granulopoiesis. Ineffective erythropoiesis causes the death of polychromatic megaloblasts.

References

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13.5 Vitamin B₆

Wolfgang Herrmann, Rima Obeid

Vitamin B₆ is the collective term for three similar, water-soluble compounds, pyridoxol/pyridoxine, pyridoxal (PL) and pyridoxamine (Fig. 13.5-1 – B₆-vitamins and their corresponding phosphates). All three forms are precursors of pyridoxal-5’-phosphate (PLP), the most active form of vitamin B₆ involved as cofactor in more than 180 enzymatic reactions in the human body. The human body itself cannot produce the cofactor pyridoxal phosphate and therefore depends on the dietary supply of its precursors.

Vitamin B₆ is ubiquitous in nature. It can be synthesized by microorganisms and also by higher plants. Vitamin B₆ is especially abundant in meat, liver, potatoes, cereals, vegetables, certain fish species (mackerel) and dairy products. About 30–40% of vitamin B₆ in animal products are lost by frying and cooking; the loss of vitamin B₆ in plant-based products by cooking is much smaller. The bio availability of vitamin B₆ ranges between 70 and 80%.

13.5.1 Indication

• Diagnosis of deficiency states
• Part of the individual risk profile for patients with degenerative cardiovascular and neurodegenerative disease

Target groups are listed in Tab. 13.5-1 – Indications for vitamin B₆ determination.

13.5.2 Method of determination

High performance liquid chromatography (HPLC)

Principle: the first step consists of sample processing and includes precipitation followed by sample derivatization. In the precipitation step, higher molecular substances are separated. The precipitate is sedimented by centrifugation and the supernatant is extracted. Vitamin B₆ is converted to a fluorescent derivative by semi carbazide or cyanide derivatization. The derivatization reagent is added simultaneously with the precipitation reagent or later (depending on the method used) and incubated for 20 minutes. After centrifugation, the supernatant is injected into the HPLC. Fractionation by HPLC is effected on a reversed-phase column using isocratic methods at 25 °C. The chromatograms is recorded by a fluorescence detector.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The LC-MS/MS method allow the simultaneous, direct and fast determination of various vitamin B₆ forms (pyridoxal 5’-phosphate, pyridoxal, 4-pyridoxic acid, pyridoxine and pyridoxamine) /1/.

Principle: plasma is deproteinized by means of trichloroacetic acid and mixed with internal standard containing Delta₂-pyridoxal-5’-phosphate, Delta₃-pyridoxal and Delta₈-riboflavin. PL, PLP, pyridoxine (PN), pyridoxine 5’-phosphate, pyridoxamine (PM), pyridoxamine-5’-phosphate, 4-pyridoxic acid (PA), riboflavin, flavin mononucleotide (FMN) and FAD are fractionated by means of a C8 reversed-phase column applying an acetonitril gradient in an acetic acid/heptafluorobutyric acid buffer. The analytes are detected and quantified using LC-MS/MS in the positive ion mode.

This method does not necessitate analyte derivatization, only requires a small sample volume, has short fractionation times (8 min. per cycle) and provides acceptable sensitivity and specificity /1/.

Enzymatic assay

Enzymatic methods are based on pyridoxal-5’-phosphate (PLP)-dependent enzymes, such as apo-tyrosine-decarboxylase, erythrocytic aspartate aminotransferase or apo-homocysteine-lyase /8/. The enzymatic reaction products are detected radioactively or chromogenically. The results of the assays show the total PLP activity of the vitamers.

Principle: the PLP assay uses the apo form of the PLP-dependent, recombinant enzyme homocysteine-α,γ-lyase (rHCYase). For apo-rHCYase extraction, PLP is removed from the holoenzyme rHCYase by incubation with hydroxylamine. Enzymatic activity is restored by holoenzyme reconstitution which is linearly correlated with the enzyme-bound PLP. By way of conversion (reconstituted holo-rHCYase) from millimolar concentrations of homocysteine to H₂S, the amplification principle of the assay allows to measure PLP concentrations in the nanomolar range. In the indicator reaction, N,N-dibutylphenylenediamine (is used as chromophor for the quantification of the formed H₂S. The enzymatic method correlates well with the chromatographic methods of determination.

13.5.3 Specimen

Serum or EDTA plasma: 1 mL

13.5.4 Reference interval

Pyridoxal-5 phosphate (PLP): ≥ 4.9 μg/L (20 nmol/L)

Method-dependent reference values of the vitamin B₆ status are shown in Tab. 13.5-2 – Methods for determining the vitamin B₆ status and their reference values. Other authors specify PLP above 30 nmol/L as normal concentrations /3/.

13.5.5 Clinical significance

The turnover time of the vitamin B₆ pool is not known in detail. It has been reported that there are two pools: a fast one with a turnover of about 0.5 days, and a slow one with a turnover of 25–33 days. The slow turnover compartment is classified as storage pool. Accordingly, vitamin B₆ can only be stored for a few weeks. Deficiency symptoms following store depletion include muscular dystrophy, skin alterations, nervous system disorders or impaired protein biosynthesis.

The determination of serum vitamin B₆ only allows limited conclusions regarding the functional, intracellular vitamin B₆ status. The detection of vitamin B₆ deficiency therefore utilizes various other available analysis methods and biomarkers that are more or less common due to their different diagnostic specificity, sensitivity and practicability.

The following analysis methods have been described for the diagnosis of vitamin B₆ deficiency:

• The most common method is the determination of pyridoxal-5’-phosphate (PLP) and pyridoxal (PL) in plasma and/or whole-blood
• Another test is to measure the excretion of xanthurenic acid following tryptophan loading. The test is based on the different response times of vitamin B₆-dependent enzymes to vitamin B₆ deficiency. Thus, urinary excretion of xanthurenic acid can be utilized as a marker of vitamin B₆ deficiency.
• Oxidation of PL by aldehyde oxidase or aldehyde dehydrogenase results in 4-pyridoxic acid. Urinary excretion of 4-pyridoxic acid can be analyzed.
• Determination of the activity of erythrocytic aspartate aminotransferase (AST) with and without PLP is also utilized as a marker. Activity is reduced in B₆ deficiency and increases after incubation with PLP.
• In homocysteine metabolism, vitamin B₆ also acts as a cofactor of two enzymes of the transsulfuration pathway, cystathionine β-synthase and γ-cystathionase. Consequently, cystathionine and homocysteine are functional biomarkers of intracellular vitamin B₆ deficiency, with cystathionine being more sensitive. However, these biomarkers are not specific of vitamin B₆ deficiency but can also be elevated in folate or vitamin B₁₂ deficiency or in renal dysfunction.

The oral methionine loading test is especially sensitive in the detection of vitamin B₆ deficiency, even at normal fasting homocysteine levels. Because, in vitamin B₆ deficiency, the transsulfuration pathway in homocysteine catabolism is only moderately impaired, the fasting homocysteine level in intact remethylation may still range within the reference interval. However, non-fasting conditions or methionine load in the setting of vitamin B₆ deficiency results in marked changes in homocysteine concentration.

13.5.5.1 Assessment of vitamin B₆-status

Metabolization analysis and pharmacokinetic studies trace the profile of all B₆ forms present. Determination of the pyridoxal-5’-phosphate (PLP) level including the precursor molecule pyridoxal (PL) has become the established method for analyzing the vitamin B₆ supply situation. PLP and PL in serum and/or plasma (and, possibly, PL in the erythrocytes) form the main B₆ vitamers available for the tissues. Impairment of the phosphorylation-based equilibrium between PLP and PL causes a change in the matrix-specific correlations of the two forms.

Since it is not always sufficient to use PLP alone as an indicator of the vitamin B₆ status, the additional use of PL is recommended /4/. In a comparison of pregnant women with a nonpregnant control cohort, PLP was 50% lower in the pregnant women, but the total amount of PLP and PL levels did not differ significantly /5/. Relating the PLP and PL concentrations to albumin did not show any differences between the two groups.

Excretion of 4-pyridoxic acid is regarded as a short-term indicator of the vitamin B₆ status. In B₆ deficiency studies, the decrease in 4-pyridoxic acid in urine was similar to that of PLP in plasma /6/. Total urinary excretion of vitamin B₆ (all forms) is not a sensitive indicator of the vitamin B₆ status.

The aminotransferase activity in the erythrocytes is considered a long-term indicator of the vitamin B₆ status /7/. The activity is analyzed with and without adding PLP. However, the reliability of this biomarker for the assessment of the vitamin B₆ status is controversial /8/.

13.5.5.2 Interactions with drugs

Treatment with certain drugs can affect the vitamin B₆ status and result in increased vitamin B₆ requirement. Tab. 13.5-3 – Interactions between vitamin B₆ and drugs shows the drugs and substances and their effects on the vitamin B₆ status /9/. A common characteristic of drug interaction is the unfavorable effect on the function of the central nervous system (CNS). Moreover, many drugs react with pyridoxal-5’-phosphate (PLP) to form a Schiff base. This reaction can cause a decrease in the PLP tissue level, which may result, for example, in dysfunction in the brain. The drugs listed below may reduce the PLP level. Therefore, an adequate supply with vitamin B₆ must be ensured upon intake of such drugs.

The following drugs cause interactions with vitamin B₆:

• Cycloserine (seromycine, tuberculosis treatment), isoniazid, (tuberculosis treatment), penicillamine (rheumatoid vasculitis, arthritis), theophylline (anti asthmatic), hydralazine (apresoline, antihypertensive). In most cases, vitamin B₆ supplementation will eliminate undesired side effects and compensate for increased requirement /10, 11/.
• Oral contraceptives do not react directly with pyridoxal-5’-phosphate (PLP), but induce the synthesis of enzymes, including some PLP-dependent ones. This results in increased PLP binding in the tissue and reduced plasma PLP concentration /12/.
• Estrogens mainly influence enzymes of the tryptophan-niacin metabolism. In some cases, the vitamin B₆ requirement can be higher in women under contraceptive treatment. Erythropoietin therapy reduces the vitamin B₆ level in the erythrocytes so that supplementation may be required /13/.
• Phenytoin (anticonvulsive) reduces the therapeutic effect of vitamin B₆ supplementation. In concurrent antibiotics therapy and vitamin B₆ supplementation, the two substances interfere with each other in intestinal absorption and should therefore not be taken at the same time.
• Levodopa. Vitamin B₆ reduces the effect of levodopa (anti-Parkinson drug), but also reduces the side effects of the treatment (vomiting, nausea). Therefore, care should be taken to ensure that the level of vitamin B₆ supplementation attenuates the side effects, but does not reduce the effect of the drug.
• Chemotherapeutics. In some chemotherapeutics like 5-fluorouracil, doxorubicin, vitamin B₆ supplementation attenuates certain side effects without reducing the effect of the drug.
• Tricyclic antidepressants. Their effect is also improved by vitamin B₆ supplementation.

13.5.5.3 Vitamin B₆-deficiency

Vitamin B₆ deficiency can develop from vitamin B₆-deficient diet, impaired vitamin B₆ metabolism or dysfunctional vitamin B₆-dependent enzymes, whose activity can partly be restored by high amount of vitamin B₆. Pronounced vitamin B₆ deficiency is rare and usually occurs as a result of other diseases. Because of the high number of vitamin B₆-dependent reactions, deficiency can manifest in many ways. The significance of pyridoxal-5’-phosphate (PLP) for numerous cellular processes and functions is based on its participation as cofactor in various enzymatic reactions (Tab. 13.5-4 – Pyridoxal phosphate-catalyzed enzymatic reactions).

Processes and their functions influenced by PLP are shown in Tab. 13.5-5 – Vitamin B₆-dependent cellular processes. Vitamin B₆ deficiency has a negative effect on humoral and cellular immunity. Vitamin B₆ treatment (50 mg/d) for 2 months improved lymphocyte responses in elderly women with impaired immunocompetence /14/.

13.5.5.4 Clinical symptoms and diseases

The most common symptoms in vitamin B₆ deficiency are peripheral polyneuropathy, vomiting, depression, impaired cognitive competence and microcytic anemia. It cannot be clearly determined in most cases whether the vitamin B₆ deficiency is based on increased catabolic rate, increased requirement, nutritional deficiency or impaired PLP synthesis. Diseases and conditions possibly associated with abnormal vitamin B₆ metabolism are listed in Tab. 13.5-6 – Abnormal vitamin B₆ metabolism, diseases and conditions.

Numerous diseases or pathological conditions are associated with abnormal vitamin B₆ metabolism. The pyridoxal-5’-phosphate (PLP) concentration or tryptophan metabolism are regarded as primary indicators in this context. However, the use of PLP alone does not provide reliable information as to whether the metabolism has actually changed. Occurrences of pellagra and anemia have been reported historically in severe vitamin B₆ deficiency. Mild vitamin B₆ deficiency has been associated with premenstrual syndrome /15/, carpal tunnel syndrome /16/ and psychiatric disease /17/. High-dose vitamin B₆ supplementation (100–200 mg/day) given to patients with carpal tunnel syndrome was found to reduce the risk of acute cardiac chest pain or myocardial infarction /18/. However, none of the above-mentioned associations are considered proven.

PLP is required for the conversion of tryptophan to niacin /3/. Diseases where tryptophan metabolism changes were determined and vitamin B₆ supplemented include asthma, diabetes, certain tumors, pellagra and rheumatoid arthritis. Diseases or conditions where low PLP concentrations have been reported include asthma, diabetes, renal disease, alcoholism, cardiac disease, pregnancy and carcinomas (Tab. 13.5-7 – Diseases associated with vitamin B₆ deficiency). Supplementation has been applied in numerous diseases or pathological conditions. High doses of pyridoxine (up to 750 mg/day) are given in enzyme dysfunction to restore normal functions. However, there is the risk that high doses of vitamin B₆ can lead to peripheral sensory neuropathies and nerve degeneration /19/.

13.5.5.5 Recommended dietary allowance

The recommended dietary allowance (RDA) of vitamin B₆ is 1.3 to 1.7 mg/day in the United States /20/ and 1.4 mg/day in Europe /21/. In 2003/4, the mean intake in the United States was 1.86 mg/day in nonusers and 1.92 mg/day in users (36% of the US population take vitamin supplements) /38/. In these cases, linear correlation between the plasma PLP level and vitamin B₆ intake was observed. Mean plasma PLP level was 41 nmol/L in U.S. males not using vitamin supplementation and 29 nmol/L in females.

Vitamin B₆ requirement also depends on protein turnover and increases as a function of protein intake because there is a positive correlation due to the involvement of pyridoxine in amino acid metabolism /39/. High protein intake induces the enzymes involved in amino acid metabolism to bind more PLP and, as a result, PLP is no longer available for other metabolic processes.

13.5.6 Comments and problems

Method of determination

The different procedures yield comparable results. However, the comparability of results obtained in different laboratories is not always satisfactory and leaves room for improving standardization /24/.

Procedures measuring one or more B₆ vitamers or the metabolite 4-pyridoxic acid are regarded as direct indices of the vitamin B₆ status. Indirect procedures determine metabolites or metabolic pathways, where pyridoxal-5’-phosphate (PLP) is essential as a cofactor and/or PLP-dependent enzyme activities are measured.

Reference interval

The reference interval for vitamin B₆ strongly depends on the nutritional state and, consequently, the selection of the subject cohort. The reference range specified for vitamin B₆ is only for orientation and should be confirmed by own data.

Stability

For determination, collect fasting blood in the morning before use of any medication.

Because vitamin B₆ is highly sensitive to light and temperature, the sample should be protected from light, cooled and centrifuged immediately. The samples are stable for 1 week if stored at 2–8 °C in the dark and remain stable for months at –20 °C. Vitamin B₆ can also be determined from whole blood. However, there are currently no generally valid reference values for this.

13.5.7 Pathophysiology

Vitamin B₆ is the collective term for three similar, water-soluble compounds, pyridoxol/pyridoxine, pyridoxal (PL) and pyridoxamine (Fig. 13.5-1 – B₆-vitamins and their corresponding phosphates). The corresponding 5’-phosphoric acid esters are the biologically active coenzymes. Chemically, they are derivatives of 4,5-bis(hydroxymethyl)-2-methylpyridine-3-ol and are distinguished by different substituents at position 4 that are involved in the coenzyme function. Having the same biological activity, the three species can be inter converted by metabolism. The following enzymes participate in the inter conversion of the different vitamers: pyridox(am)ine phosphate oxidase (PNPO, EC 1.4.3.5), pyridoxal kinase (PDXK, EC 2.7.1.35), pyridoxal phosphatase (PDXP, EC 3.1.3.74) and other phosphatases [alkaline phosphatase (ALP, EC 3.1.3.1), acidic phosphatase (ACP, EC 3.1.3.2)]. Pyridoxal is converted to 4-pyridoxate by means of aldehyde oxidase 1 (AOX1, EC 1.2.3.1). All three forms are precursors of pyridoxal-5’-phosphate (PLP), the most active form of vitamin B₆ involved as cofactor in more than 180 enzymatic reactions in the human body. The human body itself cannot produce the cofactor pyridoxal phosphate and therefore depends on the dietary supply of its precursors.

Metabolism

Vitamin B₆ is predominantly resorbed in the upper jejunum and ileum. Vitamin B₆ generated in the colon is not available to the body. Before entering the intestinal mucosa cells, the vitamers must be de phosphorylated, a process catalyzed by the membrane-bound ALP /41/. The extents to which pyridoxal-5’-phosphate (PLP) and pyridoxal (PN) are resorbed in the human body are comparable. The resorption of phosphorylated compounds is slower. The resorption of B₆ vitamers into the mucosa cells is a passive, non-saturable process. In the intestinal mucosa cell, the vitamers are re phosphorylated to PLP by pyridoxal kinase and then, before leaving the cell on the serosal side of the intestinal mucosa, de phosphorylated again. Following enteral resorption, phosphorylation to PLP and PNP takes place in the liver and other organs. Vitamin B₆ in plasma occurs as PLP (60%), PN (15%) and PL (14%). Most derivatives are bound to albumin. The binding of PLP to albumin in the circulation protects against hydrolysis and enables PLP supply to the other tissues. The protein-bound PLP (via Schiff base) is thought to form the circulating depot form of vitamin B₆ because it is membrane-impermeable and, therefore, not directly available to the cell. PLP is hydrolyzed to PL for tissue resorption by simple diffusion and, following resorption, PL is re phosphorylated again to the active coenzyme form. In the erythrocytes, PLP is mainly bound to hemoglobin, and its concentration is 4–5-fold higher than in plasma /3/. The vitamin B₆ pool in adults is estimated at 40–50 mg. Most of it is bound to glycogen phosphorylase and localized in the muscles /42/.

Biochemistry

More than 180 enzymatic reactions are PLP-dependent /43/. In enzymatic reactions, PLP is covalently bound to the corresponding enzyme via a Schiff base (Fig. 13.5-2 – Key mechanism of the transamination reaction between enzyme-bound pyridoxal-5’-phosphate and an amino acid).

PLP is involved in the metabolism of amino acids, glucose and lipids, the synthesis of neurotransmitters, histamine and hemoglobin and the function and expression of genes. The liver is the main location of vitamin B₆ metabolism. An overview of the types of PLP-catalyzed enzymatic reactions are provided in Tab. 13.5-4 – Pyridoxalphosphate-catalyzed enzymatic reactions. Reactions on the α, β and γ carbon atom of amino acids have been defined. PLP is also a cofactor in transamination, transsulfuration, deamination, decarboxylation, racemization and eliminations or replacements at the β or γ carbons /42, 43/. PLP is involved in the amino acid metabolism from synthesis to degradation: (a) Amino acid decarboxylation results in the formation of amines, which act as neurotransmitters or hormones, such as γ-aminobutyrate, histamine, noradrenaline (converted to adrenaline) or serotonin. (b) Amino acid transamination results in the formation of keto acids, which are then oxidized for metabolic energy. ( c ) A multitude of reactions pertain to the amino acid side chains, such as kynureninase (EC 3.7.1.3), cystathionine-β-synthase (EC 4.2.1.22) or γ-cystathionase (EC 4.4.1.1). (d) In phospholipid synthesis, phosphatidylserine decarboxylation results in the formation of phosphatidylethanolamine.

Besides folate and vitamin B₁₂, vitamin B₆ plays an important role as a cofactor in homocysteine catabolism. The cell toxic homocysteine stands at the intersection of two metabolic pathways: the re methylation to methionine and the transsulfuration to cysteine via cystathionine. The latter pathway is catalyzed by the enzymes cystathionine-β-synthase and γ-cystathionase, both of which need vitamin B₆ as a cofactor. Impaired homocysteine degradation can lead to hyperhomocysteinemia, which is considered an independent risk factor for vasodegenerative and neurodegenerative disease. The correlation between vitamin B₆ deficiency and postprandial increase in homocysteine has frequently been shown /44/. It has been shown in individuals with pathologic homocysteine concentration following a methionine loading test that vitamin B₆ supplementation normalized the afterload homocysteine concentration in about 50% of the individuals /45/.

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13.6 Betaine and choline

Wolfgang Herrmann, Susanne H. Kirsch, Rima Obeid

Betaine (N,N,N-Trimethylglycin)

The body’s betaine requirement is met through direct dietary intake and by oxidation of dietary choline. Betaine is abundant in cereals, especially wheat, spinach, chard and beets. Daily dietary intake of betaine is 100–300 mg. The intake differs between different populations and depends on the diet and the way the food is prepared (betaine is heat-stable). Physiologically, betaine has two functions: it acts as an osmolyte and is the source of methyl groups for numerous biochemical reactions. The differences in bio availability of betaine from different sources of food are small because betaine is strongly water-soluble and not bound to proteins. The mammalian intestine provides at least three transport systems for betaine /1/.

Choline

Choline is a water-soluble molecule often associated with the vitamin B complex. Choline is the main dietary source of methyl groups and is crucial for maintaining normal cell function and the structural integrity of cell membranes and transmembrane signaling. It has a direct influence on cholinergic neurotransmission and is required for normal muscle function and lipid transport from the liver /2/. In the prenatal period, choline is important for fetal brain development.

A choline-deficient diet causes liver injury, and choline deficiency increases the homocysteine concentration in blood, especially following methionine loading /3/. Choline deficiency can also cause developmental disorders, fetal brain injury, fatty liver or muscle injury. Moreover, deficiencies in choline, betaine or folate result in homocysteine accumulation. Hyperhomocysteinemia is an accepted risk factor for various diseases /3/. Furthermore, reduced plasma betaine concentrations have been correlated with lipid metabolism disorders, renal insufficiency and diabetes mellitus. The choline and folate metabolisms are closely interrelated. This explains the homocysteine-reducing effect of betaine and choline.

13.6.1 Indication

Homo cystinuria, pronounced hyperhomocysteinemia, alcoholic and non-alcoholic liver disease, diabetes mellitus.

13.6.2 Method of determination

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The determination of betaine, choline [and dimethylglycine (DMG)] in blood plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides maximum detection limit and test performance. Besides protein precipitation, no further derivatization steps are required for these methods.

The stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method enables concurrent measurement of betaine, choline, DMG and acetylcholine (ACh) based on simple sample processing /4/. EDTA plasma can be analyzed directly in this process.

High performance liquid chromatography (HPLC) and gas-liquid chromatography-mass spectrometry (GC-MS)

HPLC methods for betaine determination are widely used. However, these methods do not provide the sensitivity required for the quantification of serum and plasma concentrations. The first determination step using HPLC is the derivatization of betaine by alkylation of the carboxyl group to bind the functional group required for UV absorption or fluorescence. Common derivatization reagents are 2’-bromophenacyl bromide, 2’-bromophenacyl triflate and 2-naphthacyl triflate /9/.

Enzymatic methods

Enzymatic methods of betaine determination are based on reactions catalyzed by betaine-homocysteine methyl transferase (BHMT, EC 2.1.1.5) /10/. Choline determination is based on specific oxidation of choline by choline oxidase (EC 1.1.3.17) /11/. However, these methods provide little sensitivity and are time-consuming.

13.6.3 Specimen

EDTA plasma or urine: 1 mL

For (UP)LC-MS/MS methods, 100 μL of specimen are required. Betaine levels in plasma and serum are approximately identical. However, choline levels in serum are about twofold higher than in plasma (Fig. 13.6-1 – Betaine and choline levels in serum and EDTA plasma).

13.6.4 Reference interval

Tab. 13.6-1 – Fasting betaine and choline levels in EDTA plasma and urine shows guidance values for betaine, choline and DMG from the EDTA plasma and urine of several studies (LC-MS/MS method).

13.6.5 Clinical significance

Betaine and choline are important metabolites in the metabolism of lipids, neurotransmitters and amino acids. They are essential factors in homocysteine degradation and participate in the formation of S-adenosyl methionine (SAM), the universal methyl group donor. Insufficient supply can result in severe developmental disorders, fatty liver and muscle injury. Betaine and choline concentrations in plasma and urine are affected by diseases and environmental factors such as smoking and stress (Tab. 13.6-2 – Effect of diseases and lifestyle factors on plasma and urinary betaine and choline concentrations).

13.6.5.1 Clinical significance of betaine

Plasma betaine levels are remarkably stable for an extended period of time, even following significant changes in tissue concentrations. Plasma and urinary concentrations are characterized by great individuality. It has been shown that betaine concentrations are dependent on betaine intake (depending on the dose). In many cases, interpretation is exacerbated by minor changes observed under normal diet or major changes determined in supplementation studies. For this reason, plasma betaine determination is often not conclusive. Urinary betaine excretion expressed as ratio to creatinine is potentially more significant because it depends on the diet only to a small extent. Betaine levels in plasma and urine do not seem to be correlated. Urinary betaine concentrations are not correlated with age /8/. Excretion is very high in neonates, decreases within a short period of time and remains constant thereafter (betaine clearance approximately 1.5–3%). The concentration in adults seems to increase by about 1% a year. Betaine concentrations in men are about 15% higher than in women. During pregnancy, plasma betaine decreases by the same magnitude as homocysteine and related metabolites and reaches a plateau in gestation week 20 /12/.

Diseases associated with betaine deficiency are listed in Tab. 13.6-3 – Association between betaine deficiency and diseases.

13.6.5.2 Clinical significance of choline

The plasma choline status is influenced by the diet. It has been reported that choline levels were higher in individuals after a meal than in the fasting condition (Fig. 13.6-2 – Betaine and choline concentrations in EDTA plasma following overnight fasting). The marked increase in serum choline levels has been associated with a release of choline from phospholipids by serum cholinesterases (Fig. 13.6-2). These will be inhibited if EDTA, for example, is added. Choline levels are higher in men than in women and higher in older than in younger women /7/. Elevated choline concentrations (whole-blood and plasma) have been discussed as biomarkers for the risk of ischemic heart disease, particularly if no cardiac troponins are detectable on admission of the patient /13/. Choline concentrations decrease during physical activity. Several investigations have shown that plasma free choline decreases significantly in marathon runners /14/.

Choline deficiency causes the development of fatty liver and liver injury as it leads to phosphatidylcholine deficiency, which, in turn, impairs the removal of triglycerides (formed in the liver) via very-low-density lipoprotein (VLDL) /15/. Choline deficiency can also result in muscle injury with elevated creatine kinase activities /16/. Choline deficiency has also been linked to DNA damage and the death of peripheral lymphocytes /17/. Choline deficits cause hyperhomocysteinemia and increased cardiovascular risk. Moreover, choline deficiency during pregnancy results in increased risk of neural tube defects /12/. Choline deficiency during the fetal period is associated with later learning disorders or attention deficits /18/. An increased mortality risk and increased risk of breast cancer have been reported in women with low choline intake /19/. Diseases associated with choline deficiency are listed in Tab. 13.6-4 – Association between choline deficiency and diseases.

13.6.5.3 Clinical significance of N,N-dimethylglycine (DMG)

DMG is a potential biomarker for an abnormal C₁ cycle (exclusively formed via betaine-homocysteine methyl transferase). Plasma and urinary DMG concentrations show smaller intraindividual differences than betaine. DMG concentrations are elevated in folate deficiency because of increased homocysteine methylation with betaine as the methyl donor. Betaine administration results in a mild, temporary increase in plasma and urinary DMG concentrations. Clearance occurs only partly by urinary excretion. DMG is primarily cleared by the mitochondrial enzyme dimethylglycine dehydrogenase (DMGDH, EC 1.5.99.2) The DMG/betaine ratio is considered to be more conclusive than the DMG concentration because the plasma DMG concentration is partly affected by betaine intake. There is no evidence that urinary DMG concentration provides additional clinical information.

13.6.6 Comments and problems

Naturally occurring cholinesterases must be inhibited immediately after sampling. Carbamates, EDTA (Ca⁺⁺ chelating agents) or organophosphates are suited for this purpose. Choline concentration in serum is significantly higher than in plasma (Fig. 13.6-1 – Betaine and choline concentrations in serum and EDTA plasma), presumably because of the constant, serum cholinesterase-induced release of choline from phospholipids in serum. Citrate plasma has the lowest choline and betaine concentrations. EDTA plasma is recommended as specimen for betaine, choline and DMG determination.

The concentrations of betaine, choline and DMG in deep-frozen (–70 °C) plasma or urine remain stable for at least six months. Moreover, no changes in the plasma betaine, choline and DMG concentrations have been found even after several freezing-defrosting cycles.

Fasting plasma

Contrary to the choline concentration, the plasma betaine concentration is not influenced by fasting conditions. Plasma choline concentrations are higher under non-fasting than under fasting conditions. However, there is a strong correlation between fasting and non-fasting choline concentrations (Fig. 13.6-2 – Betaine and choline concentrations in EDTA plasma following overnight fasting and non-fasting). Therefore, 12 h fasting plasma is generally recommended as specimen.

Method of determination

The chemical determination of betaine is problematic because of the molecular structure. As it is strongly water-soluble, betaine is difficult to extract from aqueous solutions. An early method of determination utilized the low solubility of quaternary ammonium iodides in betaine /30/.

Betaine has a low-reactive amino group, and its likewise inactive carboxyl group is not as readily derivatized as other carboxyl compounds. Betaine derivatives present as quaternary ammonium cations can be separated using ion exchange chromatography /9/. Derivatization often also includes betaine metabolites, such as DMG. Derivatives may be formed that are alkylated both at the nitrogen and the carboxyl group, thus carrying two chromophores, are eluted too early by the silica gel column and escape reliable quantification /9/. It was not until modern chromatography/mass spectrometry procedures were introduced that significant methodological improvements were achieved.

Reference interval

No generally valid reference interval has been defined for betaine, choline and DMG in plasma or urine to date. Data from studies is either based on a small number of participants or a subpopulation. In general, however, the reference ranges of these studies coincide with each other.

Basically, the following applies to plasma and serum:

• Betaine concentrations are tendentially higher in men than in women
• DMG concentrations are below 10 μmol/L
• Choline concentrations are tendentially higher under non-fasting conditions.

Determination in 24 h urine does not provide any further clinical information because excretion is stable across the day. In random urine specimens, the concentration is related to excretion per gram (g) of creatinine.

13.6.7 Pathophysiology

Betaine

Betaine has two important roles in human physiology. One is as an osmolyte to assist cellular volume regulation. The other one is as a methyl group donor for the betaine-homocysteine methyl transferase (BHMT)-catalyzed remethylation of homocysteine to methionine.

Rat tissue betaine concentrations are higher than blood plasma concentrations in almost all organs, especially the renal medulla /39/. There are various transporters that are osmoregulated, but none are specific for betaine, including the betaine GABA transporter (BGT-1) /40/ or the carnitine receptor OCTN2 /41/. As with most osmolytes, betaine is both a compensatory and counteracting solute /42/. It enhances protein stability and is particularly effective in counteracting the denaturing effect of urea /43/.

The two functions of betaine (osmolyte and methyl group donor) interact because BHMT is osmoregulated, with high tonicity reducing its expression so that betaine metabolism (and hence the mobilization of methyl groups) decreases when high osmolyte concentrations need to be maintained.

BHMT metabolism provides about 50% of the liver’s homocysteine methylation capacity and, therefore, is of extraordinary significance for the maintenance of methionine and SAM concentrations. In the human body, BHMT is expressed in the liver, cortex of the kidney, optic lens and other cells. Mammals express a different gene product referred to as BHMT2. This designation was chosen because a gene product was identified, which did not use betaine as a substrate and where the amino acid composition shared 73% identity with the protein BHMT. Instead of betaine it uses S-methyl methionine (SMM) as a methyl donor for the methylation of homocysteine /44/.

Plasma betaine and urinary betaine excretion are determinants of plasma homocysteine /12/. The role of betaine as a methyl group donor is especially important if folate supply is limited. BHMT gene expression and activity can be increased if betaine and choline are amply available or in dietary methionine deficiency /45/. DMG, the de methylation product of betaine, inhibits BHMT.

DMG is exclusively formed via the BHMT pathway and thus reflects BHMT-mediated homocysteine methylation. Via oxidative de methylation steps, the mitochondrial enzyme DMGDH transfers the methyl group from DMG as a one-carbon unit to tetrahydrofolate, resulting in the formation of 5,10-methylenetetrahydrofolate and sarcosine. Sarcosine, in turn, can be de methylated to form glycine. On the other hand, sarcosine can be formed from glycine via the enzyme glycine-N-methyl transferase (GNMT, EC 2.1.1.20) and SAM.

Choline

Eggs and liver are important dietary sources of choline. Moreover, betaine-containing foods can also have a choline-saving effect. The average daily dietary intake of choline is 8.4 mg/kg of body weight in men and 6.7 mg/kg of body weight in women /46/. Another important source of choline is endogenous synthesis in the liver via the enzyme phosphatidylethanolamine N-methyl transferase (PEMT, EC 2.1.1.17) (Fig. 13.1-2 – Homocysteine metabolism). PEMT needs 3 SAM molecules to convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC). PC is either incorporated into the cell membrane or broken down for choline generation /2/.

Dietary choline (free or as ester) is released by pancreatic enzymes. Cholinesters include PC, phospho choline, glycerophosphocholine and sphingomyelin (SM) /2/. Choline is resorbed in the small intestine, enters portal circulation as free choline and is mainly taken up by the liver, while fat-soluble PC and SM are transported to the liver by the lymph and bypass. Therefore, the different forms of choline differ in bio availability. Choline is stored in all tissues, with uptake by the liver, kidneys, mammary glands, placenta and brain being of special significance. Choline stored in the kidney is primarily required for the formation of betaine and glycerophosphocholine. Both are organic osmolytes that promote water resorption from the renal tubule by the kidney /47/.

A small amount of choline is converted to acetylcholine (ACh) by means of choline acetyl transferase. High concentrations of choline acetyl transferase are found in the cholinergic nerve endings and in the placenta. In the brain, the availability of choline governs the rate of ACh synthesis /48/. The choline taken up by the brain may first enter a storage pool (perhaps the PC in membranes) before being converted to ACh. This pool is very important for the regeneration of cholinergic neurons and brain function. Brains of patients with Alzheimer’s disease were found to show abnormalities in phospholipid metabolism /49/.

One of the main functions of choline is the synthesis of membrane phospholipids. PC, from which SM can be formed, represents the key phospholipid class in membranes. Both PC and SM play a major role in signal transduction because they function as sources for “second messengers” /2/. Moreover, PC is also required for the formation of lipoproteins, which transport the triglycerides formed by the liver to other tissues. PC biosynthesis follows two metabolic pathways, the cytidine diphosphate-choline (CDP-choline) pathway (Kennedy pathway) and the PEMT pathway. Via the first pathway, choline is phosphorylated to form phospho choline and then converted further to CDP choline. CDP choline condenses with diacylglycerol to form PC. Alternatively, PC can be formed via the PEMT pathway using SAM as methyl donor. The PEMT pathway provides approximately 30% of the PC synthesized by the liver. Evidence of this pathway has also been provided in other tissues, including the brain and mammary glands. About 70% of the hepatic PC are formed via the CDP choline pathway. These two metabolic pathways produce different physical profiles of PC species, thus generating different signal molecules /50/.

In the inner mitochondrial membrane, choline can be oxidized to form betaine aldehyde, which is further oxidized to betaine via choline dehydrogenase (CHDH, ED 1.1.99.1) and betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). Via this pathway, choline supplies methyl groups for homocysteine re methylation. The liver and kidney are the major sites of choline oxidation. As betaine cannot be reduced to choline and choline is consumed for methylation reactions in the oxidative methylation pathway, the bio availability of choline for the alternative PC synthesis pathway is reduced.

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