6 metabolite assays in cobalamin and folate deficiency

6

Metabolite assays in cobalamin and folate deficiency

R A L P H G R E E N

Cobalamin (Cbl) and folate are essential co-factors for several key meta- boric reactions in mammalian metabolism. Deficiency of either vitamin results in interdiction of normal DNA synthesis which leads to megaloblastic anaemia. The underlying biochemical mechanisms are discussed in Chapters 1 and 2. Central to the common haematological outcome of deficiencies of Cbl and folate is the methionine synthase reaction, in which both vitamins participate. It is this reaction that leads to the formation of tetrahydrofolate, the obligate precursor of the form of folate that is required for thymidine production. Deficiency of Cbl or folate can also have other consequences presumed to relate to an interruption of other essential metabolic roles that are restricted to one of the vitamins.

Interruption of Cbl or folate-dependent reactions leads not only to a decrease in the products of those reactions, but also to an accumulation of their substrates. From a knowledge of the metabolic roles of Cbl and folate, it has been possible to predict which substrates will accumulate in Cbl or folate deficiencies. In recent years, two metabolites, methylmalonic acid (MMA) and homocysteine (HCYS) have received special attention. Improvements in analytical techniques, in particular those based on gas chromatography-mass spectrometry or high pressure liquid chromatography, have led to assays for the metabolites which have improved precision and are suitable for clinical use.

The capability to measure these metabolites in serum and urine has led to several applications which will be reviewed in this chapter. The primary utility of these tests has been for diagnosis of Cbl and folate deficiencies. Recent studies have shown that compared with direct measurement of the vitamins in the blood, metabolite assays provide improved sensitivity and specificity. Metabolite assays can therefore be used in the following set- tings: (1) for the primary diagnosis of Cbl and folate deficiencies; (2) for further testing in patients found to have subnormal or low normal blood levels of Cbl or folate; (3) to distinguish Cbl from folate deficiencies; (4) for monitoring patients' responses to treatment with Cbl or folate (either to confirm the diagnosis or to assess the adequacy of therapy).

In addition, correlation of the patterns of abnormal metabolite accumulation with clinical manifestations of disease may help to elucidate the

Baillidre ' s Clinical H a e m a t o l o g y - 533 VoL 8, No. 3, September 1995 Copyright © 1995, by Bailli~re Tindall ISBN 0-7020-2004-4 All rights of reproduction in any form reserved

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underlying biochemical mechanisms that are responsible for the clinical manifestations of Cbl and folate deficiencies. Increased levels of the metabolites MMA and HCYS may also be responsible for some of the disease manifestations associated with Cbl or folate deficiency, such as myeloneuropathy. It is also possible that the identification of a particular pattern of metabolites or the finding of some novel abnormal metabolite may shed light on the hitherto unknown metabolic functions of Cbl or folate.

Despite these numerous applications of metabolite measurement for the diagnosis of Cbl and folate deficiencies, there are still some caveats and limitations to their use. Apart from vitamin deficiencies, metabolites may also be elevated in a number of other inherited and acquired disorders. Also, because there may be alternative pathways for the conversion or degradation of some metabolites, levels are not invariably raised in deficiencies of Cbl or folate. Even when metabolite levels are raised, the degree of elevation does not always correlate with the extent of vitamin deficiency or with its clinical severity. At this time, MMA and HCYS assays are done in only a few specialized laboratories, and by several different methods. There is some variability in reported normal ranges, which is most evident in the case of HCYS. Perhaps the most significant application of metabolite measurement has been for the detection of early, subtle deficiency states of Cbl and folate including a predisposition to disease that may be associated with early subclinical deficiency of these vitamins. For example, a link has been established between elevated levels of HCYS in the blood and the risk of occlusive vascular disease and there is evidence to implicate deficiency of B-group vitamins, including Cbl and folate, in the production of hyperhomocysteinaemia, even in patients who do not show any of the typical haematological effects of these deficiencies.

COBALAMIN AND FOLATE-DEPENDENT REACTIONS AND INTERRELATIONSHIPS

The metabolic pathways that require cobalamin and those involving folate that are germane to the subject of this chapter are shown in Figure 1. Cbl is known to be required for two enzymatic reactions in humans. One, which requires adenosyl-Cbl (Ado-Cbl) is a co-factor for the enzyme methylmalonyl-CoA (MM-CoA) mutase in which MM-CoA is converted to sue- cinyl-CoA which can then enter the Kreb's cycle (Smith and Monty, 1959). Deficiency of Cbl leads to accumulation of MM-CoA and its hydrolysis product, MMA, causing a rise in plasma and urine levels of this metabolite (Cox and White, 1962; Higginbottom et al, 1978; Stabler et al, 1986). There are no known alternative metabolic pathways for conversion or degradation of MM-CoA itself, although its precursor propionyl-CoA may, if it accumulates in high enough concentrations, replace acetate in the reaction catalyzed by the enzyme citrate synthase to react with oxaloacetate and form 2-methylcitrate (Ando et al, 1972). Consequently, levels of propionate (Cox et al, 1968) and 2-methylcitrate (Allen et al, 1993b,c) may also rise in the plasma and urine.

METABOLITE ASSAYS IN COBALAMIN AND FOLATE DEFICIENCY 535

~ O - r n e t h y l e n e t e t r a h y ~

/

~ 1 SAH ~ " \ , , IIMETHYLMALONATEII- I PROP,ONATE I ~ /VAL,NE P " ' "~11 I r " ~ - I 1 " 9 " - - - - "'1 ISOLEUCINE

• / FATI'Y ACIDI I I

Figure 1. Metabolic pathways for methylmalonic acid and homocysteine involving cobalamin and folate, dU, deoxyuridine; dT, deoxythimidine; DHF, dihydrofolate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; OH Cbl, hydroxocobalamin; CH~ Cbl, methylcobalamin; AdoCbl, adenosylcobalamin.

The other enzyme that requires Cbl, methionine synthase, catalyzes the reaction between HCYS and methyltetrahydrofolate to form methionine and tetrahydrofolate (Taylor and Weissbach, 1967). Deficiency of Cbl or fotate or inadequate conversion to the forms required for this reaction (methylcobalamin (MeCbl) and tetrahydrofolate (THF), respectively) result in accumulation of HCYS in the plasma and urine (Finkelstein, 1990; Green and Jacobsen, 1995). In addition to the methionine synthase reaction, there are alternative pathways for the metabolism of HCYS. A minor reaction involves conversion of HCYS to methionine through a reaction catalzyed by the enzyme betaine homocysteine methyltransferase (Finkelstein and Martin, 1984). Both this reaction, which does not require Cbl, and the methionine synthase reaction, result in generation of methionine, referred to as the methionine cycle or the remethylation pathway for HCYS metabolism (Finkelstein, 1990; Mudd et al, 1995). The other major pathway for HCYS metabolism, which requires neither Cbl nor folate, is the transsulphuration pathway in which homocysteine is converted to cysteine in two sequential pyridoxal-dependent reactions with cystathionine as an intermediary (Finkelstein, 1990; Mudd et al, 1995). Cystathionine levels are elevated in the majority of patients with Cbl or folate deficiency (Stabler et al, 1993). The reason for this finding is obscure, since current understanding of the regulation of the methionine cycle would suggest that diminished levels of methionine and S-adenosylmethionine (AdoMet) should result in diminished activity of cystathionine 13-synthase (Finkelstein, 1990; Selhub and Miller, 1992).

In addition to the methionine synthase reaction, folate is required for a number of reactions involving the transfer of one-carbon groups in purine

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and pyrimidine synthesis as well as amino acid interconversion and catabolism (Wagner, 1995). Folate-dependent pathways proximate to the methionine synthase reaction are shown in Figure 1. Increased plasma or urine levels of several metabolic substrates of folate-dependent reactions have been described (Chanarin, 1979). These include formiminoglutamic acid (FIGLU: Zalusky and Herbert, 1962) and urocanic acid (Merritt et al, I962), products of histidine catabolism, amino-imidazole-carboxamide, an intermediary in purine synthesis (Herbert et al, 1964), formic acid (Hiatt et al, 1958), and the glycine derivates N-methylglycine and N,N-dimethylglycine (Allen et al, 1993a). With the exception of N-methylglycine and N,N-dimethylglycine, the clinical utility of tests involving measurement of these folate-dependent metabolites has limited sensitivity and specificity and in the case of FIGLU and urocanic acid requires the administration of a loading dose of histidine (Chanarin, 1979).

FACTORS INFLUENCING METABOLITE LEVELS

Methylmalonate Little is known about the factors that normally influence plasma MMA. Within cells, MMA is present in the form of its coenzyme A derivative, MM-CoA. The D-isomer of MM-CoA is produced by carboxylation of propionyl-CoA, which arises from catabolism of several amino acids, including methionine, threonine, valine and isoleucine as well as thymine, uracil, the side-chain of cholesterol and odd-carbon fatty acids (Chanarin et al, 1973; Allen et al, 1993b). The D-form is converted to L-MM-CoA by a race- mase enzyme that has no known co-factor requirements (Fenton and Rosenberg, 1995a). Only the L-form of the ester reacts with MM-CoA mutase to form succinyl-CoA (Fenton and Rosenberg, 1995a). Normal variations in food intake seem to play little role in determining plasma levels of MMA, although there is an effect of diet on urinary excretion of the metabolite (Rasmussen, 1989b). Rasmussen et al (1990a) found day-to-day variations in 33 normal subjects with a standard deviation of 31 nmol/l and weekly as well as three-monthly variations with a standard deviation of 38 nmol/1.

An additional source of MMA within the body appears to be from propionic acid produced by intestinal microorganisms (Ruppin et al, 1980) and plasma MMA levels may be lowered by bowel sterilization with antibiotics (Lindenbaum et al, 1990). Entry of MMA into the plasma requires hydrolysis of the CoA derivative, which is accomplished by a hydrolase which is specific for the D-fOrm of the ester (Kovachy et al, 1983). Apart from the Cbl-dependent isomerizeration of MM-CoA to succinyl-CoA, no other pathways for its metabolism have been described. However, injection of radiolabelled MMA into rats results in its conversion to unidentified metabolic products (Kovachy et al, 1983). Most of the metabolism of MMA is believed to be hepatic (Frenkel et al, 1974). When renal function is normal, MMA is excreted in the urine and there is a good correlation between


plasma and urine concentrations of MMA (Rasmussen et al, 1989). When renal function is impaired, urinary excretion of MMA is reduced and plasma levels of MMA rise in proportion to the serum creatinine (Rasmussen et al, 1990b; Moelby et al 1992b). Plasma concentrations of MMA are normally extremely low, in the range of 0.1-0.4 gmol/1.

In Cbl deficiency, however, levels may rise by several orders of magni- tude, attaining levels as high as 50-100 gmol/1 (Stabler et al, 1990; R. Green and D.W. Jacobsen, unpublished results). These raised levels reflect increased cellular concentrations of MM-CoA resulting from the block in MM-CoA mutase caused by lack of its essential co-factor. When levels of MM-CoA and its precursor, propionyl-CoA, rise, these compounds may serve as abnormal substrates for fatty acid synthesis in reactions that normally use malonyl-CoA or acetyl-CoA, respectively, as substrates. Although the Km of MM-CoA and propionyl-CoA for the enzymes of these reactions is much higher than the Km of the natural substrates, levels of the abnormal substrates rise in Cbl deficiency to exceed their Kin. Studies in Cbl-deficient patients (Frenkel, 1973), as well as in animals and cultured cells (Cardinale et al, 1970) have demonstrated incorporation of radiolabelled '4C propionate and 14C-MMA into odd-carbon and methyl branched-chain fatty acids (reviewed in Green and Jacobsen, 1990; Metz, 1992). Also, propionyl-CoA, when it accumulates in high enough concentrations may replace the normal substrate acetyl-CoA and undergo condensation with oxaloacetic acid in a reaction catalysed by citrate synthase to form 2-methylcitrate (Ando et al, 1972). In addition to an increase in urinary propionic acid, patients with Cbl deficiency were also reported to have increased levels of acetic acid in their urine (Cox et al, 1968). This may result from metabolism of propionate to acrylate and then through one of two alternative pathways (lactate or mal- onate) to acetate (reviewed in Fenton and Rosenberg, 1995a). The metabolic shunt through propionate and MMA to succinate has been long considered to be a metabolic curiosity of no biological importance. Allen et al (1993b) however, suggest that intermediates in this pathway may have some undiscovered biochemical role.

The major usefulness of MMA measurement either in the plasma or the urine is for the diagnosis of Cbl deficiency. Cbl deficiency can arise from rare dietary lack, in vegans, or from malabsorption caused by failure of either the gastric or ileal phases of Cbl absorption, or from the chemical inactivation of Cbl. Among patients with clinically significant Cbl deficiency, pernicious anaemia is the most common cause (Chanarin, 1979). Cbl deficiency is particularly prevalent among the elderly 0~lsborg et al, 1976; Magnus et al, 1982; Blundell et al, 1985; Nilsson-Ehle et al, 1989; Yao et al, 1992; Pennypacker et al, 1992; Groene et al, 1995). This subject is reviewed in Chapter 12.

Homocysteine HCYS is derived almost exclusively from dietary methionine as there are only trace amounts of HCYS present in foods. Dietary composition appears to play little role in determining plasma HCYS in normal individuals,

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although any possible relationship to methionine intake is complicated by differences in the dietary intake of folate, Cbl and pyridoxine, the vitamin co-factors involved in metabolism of HCYS. Remethylation of HCYS tba-ough the methionine cycle and transsulphuration to homocysteine, the two major metabolic pathways for HCYS, are tightly regulated by intermediates at several points in the pathways (Finkelstein, 1990; SeLhub and Miller, 1992). Changes in activity or disturbances in a step of one of the HCYS metabolic pathways has a regulatory effect on other steps in that pathway as well as on the other pathway. For example, AdoMet, the first product of the methionine cycle, affects a key enzyme in the transsulphuration pathway, cystathionine ~-synthase (CBS) as well as two enzymes involved in the methionine cycle, methylene tetrahydrofolate reductase (MeTHFR) and betaine:homocysteine methyltransferase (BHMT). AdoMet increases CBS activity by allosteric activation so that HCYS is diverted to the transsulphuration pathway. Conversely, AdoMet decreases remethylation of HCYS by inhibiting both MeTHFR and BHMT (Kutzbach and Stokstad, 1971; Finkelstein and Martin, 1984). The subject is more extensively reviewed by Finkelstein (1990) and Mudd et al (1995). Other intermediates in these pathways such as S-adenosylhomocysteine also influence the metabolic pathways. Several physiological factors are known to influence circulating HCYS concentrations (Ueland et al, 1993; Jacobsen et al, 1994). Age and gender influence plasma HCYS concentration. Homo- cysteine levels appear to rise with increasing age, but since plasma HCYS varies inversely with vitamin nutritional status, it is possible that the age effect may be mediated, at least in part, through differences in nutritional status. In general, among adults, HCYS levels are higher in men than in women. The basis is probably hormonal, since gender differences decrease after the menopause. The serum HCYS concentration is significantly higher than that of the plasma. This is believed to result from HCYS release from formed elements when blood is allowed to clot. It is unclear what role the kidney plays in HCYS homeostasis. This has been reviewed recently (Green and Jacobsen, 1995). Although the renal excretion of HCYS accounts for only 1% of daily HCYS production (Mudd and Poole, 1975), plasma HCYS levels rise in chronic renal disease (Wilcken et al, 1981). Elevated plasma levels of HCYS have been reported in hypothyroidism (Chong et al, 1993). It is not known how thyroid function influences HCYS metabolism. Elevated plasma levels of HCYS have been reported in alcoholics (Hultberg et al, 1993b). Folate nutrition and metabolism is, however, impaired in alcoholics (Eichner and Hillman, 1971; Savage and Lindenbaum, 1986). There is also evidence that pyridoxine metabolism is impaired by chronic alcohol abuse (Lumeng and Li, 1974). The effects of alcohol on HCYS levels may therefore be secondary to changes in folate and pyridoxine metabolism.

Homocysteine levels rise in deficiencies of folate, Cbl and pyridoxine as well as in several inboru errors of metabolism affecting enzymes in the transsulphuration and remethylation pathways of HCYS metabolism. These include CBS deficiency, MeTHFR deficiency and various mutants affecting the processing of the Cbl coenzyme that is required for the methionine syn-


thase reaction. These have been extensively reviewed by Mudd et al (1995) and by Fenton and Rosenberg (1995b). In some patients who show no obvi- ous stigmata of inborn errors of HCYS metabolism, plasma HCYS may be elevated above normal in the absence of any objective evidence of folate, Cbl or pyridoxine deficiency, renal disease or other known causes of hyperhomocysteinaemia. These patients may represent heterozygotes for defective enzymes in the remethylation or transsulphuration pathways for HCYS metabolism. A substantial number of patients known to be obligate bet- erozygotes for CBS deficiency have been found to have elevated plasma levels of HCYS (Boers et al, 1985; Clarke et al, 1991). There are two major clinical uses for HCYS measurement; on its own, as a risk factor for occlusive vascular disease and together with MMA, for diagnosis and differenti- ation of Cbl and folate deficiencies. Folate deficiency results in an increase in serum HCYS (Kang et al, 1987; Stabler et al, 1988). Folate deficiency is usually nutritional in origin and is particularly prevalent among the poor, alcoholics and file elderly, as well as during pregnancy. Less commonly, folate deficiency is caused by malabsorption, drugs and increased utiliza- tion that results from increased cell turnover (Chanarin, 1979).

Other metabolites

In addition to MMA and HCYS, levels of other metabolites may be affected in Cbl or folate deficiency. Some metabolites lie directly upstream of the metabolic steps that are blocked in Cbl or folate deficiency, whereas others arise through metabolic overflow via alternative metabolic pathways. In the MMA pathway, examples of the former type include propionate, and of the latter type methylcitrate and acetate. In the HCYS pathway, upstream compounds include methionine and the overflow metabolites include cystathionine and N-methylglycine (Allen et al, 1993b). The accumulation of metabolites other than MMA and HCYS in Cbl and folate deficiencies is of interest, but is of limited practical value and appears to add little beyond the sensitivity and specificity that can be attained with serum MNL& and HCYS alone.

DETECTION METHODS

Plasma concentrations of the various metabolites that accumulate in Cbl and folate deficiencies are normally low, and generally in the nanomolar to micromolar range. Consequently, early methods for detection of these metabolites lacked the sensitivity and precision necessary for the development of reliable clinical assays. To design suitable methods for measurement of MMA and HCYS, it was necessary to overcome several technical problems. MMA is only one of several dicarboxylic acids present in the urine and plasma. It is therefore necessary to separate these potentially interfering compounds or their products before or after derivatization or ionization of these compounds. Regarding HCYS, most techniques make use of derivatization with the sulphhydryl group of HCYS. It is therefore

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necessary to ensure that HCYS is released from its association with other thiol groups by reduction, and also to distinguish HCYS from other low molecular weight thiols such as cysteine, cysteinylglycine and glutathione following derivatization. Cysteine is normally present in plasma in concentrations that far exceed that of HCYS. Earlier methods were based on colorimetric measurements, thin layer or gas chromatography.

The separation techniques of gas chromatography-mass spectrometry (GC-MS) and high pressure liquid chromatography (HPLC) coupled with improved, sensitive detection techniques such as selected-ion monitoring (for GC-MS) and fluorescence detection (for HPLC) as well as rapid, solid- phase sample clean-up and preparation have satisfied the requirements for acceptable clinical assays for MMA and HCYS, including ease and speed of analysis, sensitivity, accuracy and good reproducibility (Marcell et al, 1985; Rasmussen, 1989a; Ueland et al, 1993).

Methyimalonic acid Urine concentrations of MMA are higher than those in plasma (Higginbottom et al, 1978; Marcell et al, 1985; Rasmussen, 1989b). Consequently, early attempts to measure the accumulation of MMA in Cbl deficiency were directed to measurement of this metabolite in the urine (Cox and White, 1962; Giorgio and Plaut, 1965; Bashir et al, 1966; Brozovic et al, 1967; Gompertz, 1968; Chanarin et al, 1973; Frenkel and Kitchens, 1977). These methods, which were based either on colorimetric, chromatographic or enzymatic techniques gave considerable overlap in urinary excretion of MMA between Cbl-deficient patients and normals. The distinction was improved following the administration of an oral loading dose of L-valine or L-isoleucine, but Chanarin et al (1973) reported that a substantial number of patients with untreated pernicious anaemia (6 out of 23) had normal MMA excretion following the administration of 10 g of L- valine. In light of information that has come from more recent studies using GC-MS, such findings were almost certainly attributable to methodological problems. Norman et al (1979) described a GC-MS method for measuring urine MMA by selected-ion monitoring following cyclohexanol derivatization of MMA without extraction or purification. Later methods included a solvent extraction step for partial purification of MMA (Norman et al, 1982; Matchar et al, I987). With these improved methods, it was possible to define a normal range for urine MMA and to show increased levels of urinary MMA in patients with Cbl deficiency (Norman et al, 1982; Matchar et al, 1987).

The first published description of a method to measure MMA in plasma using selected-ion monitoring GC-MS used tert-butyldimethylsilyl derivatization of MMA following plasma extraction and purification (Marcell et al, 1985). Sample pre-treatment involved several extraction steps followed by HPLC purification. Later modification of technique by this group, involving simplification of the extraction and solid-phase purification, resulted in shorter total assay time, improved assay performance and a lower normal range (Allen et al, 1990; Savage et al, 1994a). An alternative


method for plasma MMA measurement by GC-MS was described by Rasmussen (1989a) using the dycyclohexyl derivative of MMA. Both methods use the deuterated stable isotope of MMA as an internal standard. The normal ranges for serum MMA measured by GC-MS were originally as follows: 160-640nmol/1 (Marcell et al, 1985); 80-560nmol/1 (Rasmussen, 1989a); < 800nmol/1 (DW Jacobsen and R Green, unpublished results). With progressive improvements in published methodology by Allen's group and by Rasmussen, as well as by our own laboratory and others there has been a decrease in reported normal reference ranges for serum MMA which, based on the mean + 3 SD after log transformation to correct for skewing at higher values are now in good agreement. These are as follows: 53-376 nmol/1 (Allen et al, 1990); 50-440 nmol/1 (Rasmussen and Nathan, 1990); 79-376nmol/1 (DW Jacobsen and R Green, unpublished results); < 400 nmol/1, using the cut-point for 95% of the normal population, (B Gilfix, personal communication). A GC-MS method has also been described for MMA and other organic acids that is suitable for urine and plasma, and is designed for screening of inborn errors of metabolism. It uses 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine HC1 to derivatize oxoacids followed by liquid partition chromatography (Hoffmann et al, 1989). Use of this method for measuring MMA levels in Cbl and folate deficiencies has not been reported.

Recently, methods have been described for measurement of serum MMA by HPLC (Schneede and Ueland, 1993; Babidge and Babidge, 1994) and by capillary electrophoresis (CE: Schneede and Ueland, 1995). In both methods developed by Schneede and Ueland, MMA is derivatized using the fluorescent compound 1-pyrenyldiazomethane to yield a fluorescent monoester adduct. After separation by HPLC or CE, short-chain dicarboxylic acids are quantified following laser-induced fluorescence activation. Results obtained with the CE method showed good correlation when compared to those obtained using GC-MS methods described by Stabler et al (1986), and by Rasmussen (1989a). In the alternative HPLC method described by Babidge and Babidge (1994) MMA is derivatized using monodansylcadaverine and dicyclohexylcarbodiimide. This assay was developed for veterinary use and has not been applied to measurement of MMA in human serum.

Either serum or urine MMA measurements provide similar information regarding the pertubations in MMA metabolism that occur in Cbl deficiency. Compared with measurement of urine MMA, however, serum assays offer several practical advantages. First, when urine MMA is measured, it is necessary to carry out either timed (24 hour) urine collection or, if a random spot urine is used, then to measure creatinine and express the result in terms of the creatinine concentration to make allowance for variations in urine dilution (Norman et al, 1982). Measurement of serum MMA offers the added convenience that the same specimen may be used as that submitted for serum Cbl measurement. Furthermore, urine MMA is influenced by food intake, whereas serum MMA is only minimally affected by diet (Rasmussen, 1989b). On the other hand, serum MMA levels do rise in renal failure (Rasmussen et al, 1990b; Moelby et al,

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1992b), whereas urine MMA, normalized for urine creatinine, corrects for variations in renal function and for dehydration (Norman et al, 1982; Norman and Morrison, 1993). Since Cbl deficiency is more prevalent among the elderly, a group in whom kidney disease is quite common, measurement of urine MMA offers the theoretical advantage that the con- founding variable of impaired renal function does not influence the assessment of Cbl status.

Comparison between urine and serum MMA measurement

Norman has pioneered the application of GC-MS for detection of clinical Cbl deficiency by using this technique to measure urine MMA (Norman et al, 1979, 1982). Using this assay, Matchar et al (1987) studied a group of patients who had serum Cbl assays. All 7 of 96 evaluable patients who were judged to be clinically Cbl-deficient on the basis of low serum Cbl, blood count or smear findings or abnormal Schilling test results had urine MMA levels > 5 [tg/mg ereatinine. Only 1 of the 89 patients not judged to be clinically Cbl-deficient had elevated urine MMA. On the basis of this limited study, the authors calculated a sensitivity of 100% and a specificity of 99% for the urine MMA assay. Subsequently, groups of elderly non-anaemic persons who visited a health fair and a group living in retirement apart- ments were screened using urine MMA (Norman and Morrison, 1993). Elevated levels were reported in 3% and 5.1% of the two groups, respectively. Approximately one-half of the individuals with elevated MMA levels had normal serum Cbl concentrations. Serum MMA levels were elevated in 15 out of 16 subjects who had elevated urine MMA levels. In view of this good correlation between serum and urine MMA, it is not clear why the prevalence of deficiency in the study by Norman and Morrison (1993) is substantially lower than that reported by other investigators using serum MMA (Pennypacker et al, 1992; Groene et al, 1995). More extensive com- parisons between serum and urine MMA need to be carried out in order to resolve this discrepancy. Using the same method for urine MMA, 10 out of 79 patients with elevated levels were found to have Cbl deficiency (Chao- Hung et al, 1987). Nine of the ten patients were found to have serum Cbl concentrations of < 75 pg/ml and the remaining patient had a serum Cbl level of 272 pg/ml. All patients had marked megaloblastic changes in their bone marrows with elevated serum lactate dehydrogenase. In the remaining 69 patients, urine MMA was normal and there was no evidence of megaloblastic haematopoiesis. Serum Cbl levels were reported to be 'essentially normal' but details were not provided. Stabler et al (1991) have measured MMA in the cerebral spinal fluid (CSF) and found that the concentration of this metabolite was higher in the CSF than in the serum, on average approximately two to three-fold. These workers showed a marked increase in levels of MMA in the CSF in three patients who had neuropsychiatric syndromes caused by Cbl deficiency as well as in one patient who abused nitrous oxide and had a normal serum Cbl level. The level of CSF MMA in these patients was approximately 600 times greater than normal. Of interest, was the observation that MMA levels in CSF were not increased in five


out of six patients who had elevated serum MMA levels associated with renal failure.

Homocysteine HCYS can be detected in the plasma and the urine. Assays for urine HCYS are not in clinical use except for detection of homocystinuria (Mudd et al, 1995). In the plasma, most HCYS is present in the form of the oxidized homodimer homocystine, or as mixed disulphides of HCYS, either with free cysteine or with protein-cysteinyl residues. Less than 5% of the total plasma HCYS is present in the free reduced form (Mansoor et al, 1992). To measure the total plasma HCYS, it is therefore necessary to precipitate protein and reduce disulphide bonds. In most methods, with the exception of those based on electrochemical detection and radio-enzymatic assay, the SH-group of HCYS is then derivatized using a thiol-specific reagent and the resulting adduct detected. Methods for measurement of total plasma or serum HCYS have been extensively reviewed (Ueland et al, 1993). Most of these methods give comparable results and reported normal ranges are similar (Ueland et al, 1993). Some methods allow for the simultaneous determination of other metabolites that may be clinically useful. Thus, HPLC methods that use a fluorochromophore have the capability to measure all free plasma thiols, which are separated by HPLC prior to detection (Jacobsen et al, 1989). When HCYS is measured by GC-MS, several other related metabolites can also be quantified (Allen et al, 1993b).

Loading studies

By administering a loading dose of the precursor of a particular metabolite, it may be possible to accentuate a borderline or equivocal abnormality in a metabolic pathway. In general, this approach has been used to amplify urinary metabolite excretion abnormalities, and examples include histidine loading for FIGLU and urocanic acid excretion and L-valine or L-isoleucine loading for MMA excretion (Chanarin, 1979). The effect of an L-isoleucine load on serum MMA levels has been reported in normal subjects by Rasmussen (1989b), who showed that following 100 mmol L-isoleucine, an average increase in serum MMA of 0.072 mmol/1 was decreased to 0.013 mmol/1 when the test was repeated following administration of Cbl. L-isoleucine loading has also been used to amplify the impairment of MMA metabolism that occurs in Cbl-deficient patients (Moelby et al, 1992a).

Oral methionine loading (100mg/kg body weight) has been used to accentuate abnormalities in HCYS metabolic pathways. Peak plasma levels are usually attained 6-8 hours following the oral loading dose (Sardharwalla et al, 1974; Andersson et al, 1992). The main application of the methionine loading test has been for detection of heterozygosity for cystathionine l]-synthase deficiency. Defects in this enzyme, which cataly- ses the first step in the transsulphuration pathway for HCYS metabolism, account for the vast majority of cases of the inherited disorder homocystinuria (Mudd et al, 1995). McGill et al (1990) have concluded that there

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is considerable overlap between the results of methionine loading studies in obligate heterozygotes for cystathionine 13-synthase deficiency and normal individuals. Detailed studies on the possible usefulness of the methionine loading test for diagnosis of Cbl or folate deficiencies have not been carried out, but it is of interest that raised basal levels of HCYS respond better to supplemental folic acid than to pyridoxine, whereas abnormalities in the post-load HCYS respond better to supplemental pyridoxine (Boers et al, 1985). Typically the amino acids used for these studies have an unpleasant, bitter and metallic taste, and since loading studies do not appear to provide any advantage over basal measurements of MMA and HCYS in the plasma for diagnosis of Cbl or folate deficiency states, there does not appear to be any place for the use of these tests in routine diagnosis at this time.

LIMITATIONS OF STANDARD LABORATORY TESTS FOR THE DIAGNOSIS OF FOLATE AND COBALAMIN DEFICIENCIES

Haematologicai measurement

As methods for the diagnosis of Cbl and folate deficiencies have become more sophisticated, patients who lack the classical textbook features of these deficiencies have becomes increasingly recognized. Carmel (1988) noted the absence of anaemia in 19% and the absence of macrocytosis in 33% of 70 patients with confirmed untreated pernicious anaemia who were initially screened on the basis of serum Cbl levels < 200 pg/ml. Reasons for masking of the typical haematological features of Cbl and folate deficiencies remain to be fully elucidated. Masking of macrocytosis may be due to the presence of a coexisting macrocytic process including thalassaemia minor, iron deficiency and the anaemia of chronic disease (Solanki et al, 1981; Green et al, 1982; Spivak, 1982). With respect to Cbl deficiency, patients with predominantly or exclusively neurological manifestations are being recognized in increasing numbers (Lindenbaum et al, 1988; Healton et al, 1991). The curious absence of anaemia in patients with pernicious anaemia who present with neurological features was first observed over 100 years ago (Minnich, 1892), and subsequently, by several other authors (Waters and Mollin, 1963; Chanarin, 1979). There has been renewed interest in this observation, with the advent of improved methods for diagnosis and monitoring of Cbl deficiency (Carmel, 1988, 1990; Green, 1994). Lindenbaum et al (1988) observed that 40 out of 141 patients with neuropsychiatric disorders attributable to Cbl deficiency (28.4%) had absence of anaemia or macrocytosis and in 19 of these (13.5%) both the haematocrit and mean cell volume were normal. In a more detailed and extensive study, this group also noted that less anaemic patients with Cbl deficiency tended to have more prominent neurological involvement (Healton et al, 1991). The reason for this inverse relationship has not been fully elucidated, although some circumstantial evidence points to a possible role for folate. It has long been known that treatment with folic acid can ameliorate the


haematological but not the neurological manifestations of Cbl deficiency (Schwartz et al, 1950). A recent study on Cbl-deficient patients from Zimbabwe provides some support for this hypothesis. Patients with normal or increased serum folate levels more commonly displayed neurological signs than those with low serum folate concentrations (Savage et al, 1994b).

Serum cobalamin assay

Assays for serum Cbl and folate have, for some time, constituted the standard screening test for diagnosis of deficiency of these vitamins. Previously considered to be sensitive screening tests for the detection of Cbl and folate deficiencies, there is now a mounting body of evidence to indicate that clinically significant deficiencies can occur with only slightly lowered serum vitamin levels. Indeed they can occur even in the face of serum levels that arc within the normal range. It has been estimated that of all patients with confirmed Cbl deficiency, 90-95% have serum Cbl levels < 200pg/ml, 5-10% have levels of 20(0300 pg/ml and up to 1% may have values greater than 300pg/ml (Lindenbaum et al, 1990; Stabler ct al, 1990). In several recent studies on elderly subjects, objective evidence of Cbl deficiency has been found in patients with serum Cbl levels of 200-300 pg/ml, which is at the low end of the normal range (Berlinger, 1991; Pennypacker ct al, 1992; Yao et al, 1992; Groene et al, 1995). Lindenbaum et al (1990) found a normal serum Cbl level in 9 out of 173 patients with documented Cbl deficiency. The finding of a normal serum Cbl level in deficient patients may be more frequent in elderly patients (Berlinger, 1991). Yao et al (1992) studied 100 geriatric subjects and found serum Cbl levels of < 200 pg/ml in 16%, as well as 21% with serum CbI levels of 201-299 pg/ml. Among the group studied by Yao et al (1992), MMA and/or HCYS levels were raised in four out of five with serum Cbl levels of < 200 pg/ml and in three out of nine with serum Cbl levels of 201-299 pg/ml. In the study by Pennypacker et al (1992), the frequency of deficiency as judged by elevated metabolite levels was similar, among patients with serum Cbl levels in the low normal range of 201-300 pg/ml (56%), to what it was in patients with low serum Cbl levels of _< 200 pg/ml (62%). In addition, these workers found elevated metabolites in 10 out of their 152 patients (7%) who had serum Cbl levels of > 300 pg/ml. It is not known why some patients with Cbl deficiency may have normal serum Cbl levels. The distribution of Cbl between its plasma binding proteins may play some role. Most of the plasma Cbl is bound to transcobalamin I (TCI), one of the family of Cbl-binding proteins known as R-binders or haptocorrins (reviewed in Jacob et al, 1980; Feruandes-Costa and Metz, 1982). The exact function of this Cbl-binding protein is unknown, but it plays little or no role in cellular delivery of Cbl. Most of the remaining minor fraction of plasma Cbl (10-20%) is bound to transcobalamin II (TCII). TCII-bound Cbl is rapidly cleared from the plasma (Hall and Finkler, 1965) through cellular removal by receptor- mediated endocytosis (Youngdahl-Turner et al, 1978). It has therefore been proposed that TCII-bound Cbl represents the functional and clinically

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important fraction of circulating Cbl that has been termed holo-TCII. There is some evidence to support the concept that holo-TCII measurement may provide a better index of Cbl status (Herzlich and Herbert, 1988; Goh et al, 1991). Several observations support the concept that a low level of holo- TCII is the critical factor that determines cellular Cbl delivery. Patients with congenital deficiency of TCII manifest features of severe Cbl deficiency, yet they usually have normal or near normal serum Cbl levels (Hakami et al, 1971; Cooper and Rosenblatt, 1987). Cbl deficiency associated with low levels of holo-TCII but occurring in conjunction with increased amounts of Cbl bound to the TCI fraction might go undetected if only the total serum Cbl is measured. For example, plasma R-binders orig- inate in granulocytes, and because levels rise in myeloproliferative disorders, rare patients have been described with coexistent chronic myeloid leukaemia and pernicious anaemia in whom serum Cbl levels appear normal (Corcino et al, 1971; Jacobson and Green, 1978). Normal serum Cbl concentrations in patients with Cbl deficiency have also been described in patients with intestinal bacterial overgrowth (Murphy et al, 1986) and in patients with Cbl deficiency caused by nitrous oxide (Amess et al, 1978; Layzer, 1978). Lindenbaum et al (1990) reviewed the records of 419 patients with clinically significant Cbl deficiency and found 12 in whom the serum Cbl was greater than 200 pg/ml. Although anaemia was usually absent or mild, five patients had neurological complications that responded to Cbl treatment. Three of the patients had serum Cbl levels of > 300 pg/ml; all had elevated levels of serum MMA and HCYS. The 12 patients with normal serum Cbl comprised 2.9% of the 419 patients with clinical Cbl deficiency seen over a 13 year period. However, these authors point out that 9 of the 12 patients were seen during the last 4 years of the observation period, when they were more aware of atypical presentations of Cbl deficiency. These nine patients constituted 5.2% of the 173 seen during that period.

The serum Cbl assay also lacks specificity. A serum Cbl level of less than 100pg/ml usually signifies Cbl deficiency. Levels of 100-200 pg/mt are, however, not specific for Cbl deficiency. For reasons that are not clear, a large percentage of patients with folate deficiency have subnormal serum Cbl concentrations (Chanarin, 1979). In a study of 142 patients with folate- deficient megaloblastic anaemia, Mollin et al (1962) reported serum Cbl concentrations of < 180pg/ml in almost half of 142 patients with folate- deficient megaloblastic anaemia. In 15 of these patients the level was below 100 pg/ml. Another known cause of subnormal serum Cbl levels not associated with Cbl deficiency p e r se is congenital deficiency of the R-binding proteins. R-binder (TCI) deficiency is not associated with clinical Cbl deficiency, presumably because the TCII-associated fraction of plasma Cbl is normal. Since the major fraction of plasma Cbl is normally associated with TCI, the serum Cbl concentration in TCI-deficient individuals is below normal (Cooper and Rosenblatt, 1987). In most situations in which there is a low serum Cbl level without other laboratory evidence or objective clinical evidence of Cbl deficiency, there is no apparent explanation for this finding (Brynskov et al, 1983). Unexplained low serum Cbl levels are fre-


quently found in patients with food Cbl malabsorption (Carmel et al, 1988). It is not known whether the serum Cbl level in heterozygous carders for TCI deficiency are low. The finding of an isolated low serum Cbl level is probably most often due to overlap between the lower end of the serum Cbl distribution in the population with the range considered to be deficient. It has been estimated that up to one-half of patients with subnormal serum Cbl levels do not have actual Cbl deficiency (Green et al, 1990; Stabler et al, 1990; Moelby et al, 1990; Savage et al, 1994a). Conversely, almost half of all patients with clinically confirmed Cbl deficiency have serum Cbl levels in the 100-200 pg/ml range (Allen et al, 1990). Carmel (1988) found this to be the case in 25 out of 70 patients (36%) with pernicious anaemia. Therefore, any attempt to improve specificity by lowering the cutoff for deficiency would result in a marked loss of sensitivity.

Serum and red cell folate assay

There are several problems with the sensitivity of serum and red cell folate assays. Although many of these are methodological, others relate to a failure of these measurements to consistently reflect underlying tissue folate deficiency. Many of the technical problems stem from methodological changes that have occurred during the past 30 years since folate assays were first developed. The original microbiological assays have been replaced by radioligand binding assays. The majority of these assays are performed using commercial radio-assay kits, many of which are inadequately controlled and standardized, particularly for red cell folate assay. Recently, faulty calibration of a radio-assay folate kit which resulted in a 30% overestimation of the measured folate (resulting in under reporting of folate deficiency), has been described (Levine, 1993; FASEB Report, 1994). Another technical problem that may cause spuriously elevated serum folate measurements is haemolysis. Because the concentration of folate in red cells is more than 100-fold greater than in the serum, even mild degrees of haemolysis can give rise to spurious elevation of the serum folate. There is a widely held view that recent food consumption can cause the serum folate to rise sufficiently to obscure a possible underlying deficiency. Most of the evidence, however, indicates no such effect (Reizenstein, 1965; Chanarin, 1979). For reasons that are not understood, the serum folate concentration may lie within the normal range in some patients with unequivocal evidence of folate deficiency (Eichner et al, 1972; Savage et al, 1994a; Lindenbanm and Allen, 1995).

In addition to problems with the sensitivity of the serum folate assay, it also lacks specificity. A low serum folate concentration is not always indicative of deficiency. As pointed out by Allen et al (1990) this is due in part to the manner in which the normal range is constructed, and the fact that 2.5% of normal healthy individuals may be expected to have levels that are less than the cutoff point 2sD below the mean. When the dietary intake of folate ceases or is markedly reduced, the serum folate level falls and may reach the deficient range within a few days (Herbert, 1962). Low serum folate concentration without actual evidence of deficiency has also been

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described in pregnant women and in patients receiving anti-convulsants (Chanarin et al, 1968; Lindenbaum and Allen, 1995) and also following ingestion of large amounts of alcohol (Eichner and Hillman, 1971). The serum folate concentration is also influenced by Cbl status, with serum folate levels being above normal in approximately 20% and below normal in 10% of patients with Cbl deficiency (reviewed in Chanarin, 1979).

On theoretical grounds, measurement of the red cell folate concentration should provide a more reliable assessment of cellular folate status than the serum folate (Hoffbrand et al, 1966). However, this test also lacks sensitivity and specificity. In patients with megaloblastic anaemia caused by folate deficiency during pregnancy, red cell folate measurements were normal in 26% (Varadi et al, 1966) and 11% (Chanarin et al, 1968) of individuals. In alcoholics, red cell folate measurements were normal in 61% (Hines, 1969) and 31% (Savage and Lindenbaum, 1986) of patients. Patients with mild to moderate folate deficiency and normal red cell folate have also been reported by Jones et al (1979) and by Bain et al (1984). There is a major difficulty with the specificity of a low red cell folate concentration because it is frequently caused by Cbl deficiency. Over half of patients with pernicious anaemia have low red cell folate concentrations (Chanarin, 1979). There are even greater concerns regarding the validation and reliability of red cell folate assay kits. Apart from one report (Bain et al, 1984) red cell folate kit assays have not been studied adequately.

In isolation, serum or red cell folate measurements are of limited value. In conjunction with a serum Cbl level, more useful information may be obtained. Since most radio-assay kits are designed to measure serum Cbl and folate simultaneously, it was previously the case that there was no additional expense involved in obtaining both measurements together. However, there has been a recent major trend away from radio-assays to non-isotopic methods based on enzyme linked spectrophotometric and chemiluminescent detection techniques. Semi-automated instruments designed for random access are used for these assays so that individual, rather than simultaneous measurements for serum Cbl and serum folate are carried out.

CLINICAL STUDIES IN PATIENTS WITH COBALAMIN AND FOLATE DEFICIENCIES

Historically, interest in the clinical measurement of MMA and HCYS first arose as a means to diagnose rare inborn errors of metabolism affecting the pathways involved in the conversion and metabolism of these compounds. The syndrome of mental retardation, multiple congenital abnormalities and precocious thromboembolism in association with high levels of homocystine in the urine gave rise to the description of the syndrome of homocystinuria and led shortly thereafter to the identification of the underlying inherited metabolic defects that could produce this syndrome including CBS deficiency, a Cbl processing defect and MeTHFR deficiency (reviewed in Green and Jacobsen, 1995; Mudd et al, 1995). Similarly, the

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demonstration of methylmalonic aciduria in acidotic infants with failure to thrive led to the description of several mutants affecting either the methylmalonyl CoA isomerase enzyme or the generation of 5'-adenosyl-Cbl (reviewed in Cooper and Rosenblatt, 1987; Fenton and Rosenberg, 1995a). An improved understanding of the underlying metabolic pathways involved in MMA and HCYS metabolism, including the specific co-factor requirements for enzymes involved in these pathways, stimulated an interest in the potential utility of measuring these metabolites in the urine of patients with nutritional and other acquired vitamin co-factor deficiencies. The improvements and refinements of these methods then also led to assays for measurement of the metabolites in plasma. In the case of HCYS, a major impetus for the development of clinical assays to measure HCYS came from the suggestion that raised plasma levels of this metabolite were associated with an increased risk of vascular disease (McCully and Wilson, 1975).

Currently, the major applications of clinical assays for the metabolites MMA and HCYS are for the diagnosis of deficiencies of Cbl or folate. Clinically significant deficiencies of Cbl or folate occur when cellular levels are inadequate to satisfy the co-factor or substrate requirements for these vitamins. This interferes with key biochemical pathways and leads ultimately to the disease manifestations found in patients with Cbl and folate deficiencies. Detection of the metabolic disturbances that accompany vitamin deficiency and that often precede actual disease manifestations offers the possibility of providing an early clue to diagnosis, and a means for early recognition of Cbl and folate deficiency states. Accurate direct determinations of Cbl and folate status are not provided by measurements of serum levels of these vitamins. There are several possible reasons for this which have been addressed above. For two main reasons, more attention was directed, at first, to the development of assays for MMA than HCYS for the diagnosis of Cbl or folate deficiencies. First, on theoretical grounds, it was considered that accumulation of MMA might offer a more specific test for Cbl deficiency because of the exclusive co-factor requirement for Cbl (and not folate) in the metabolism of MMA. Second, unlike MMA which has no alternative pathway for its conversion or degradation other than the Cbl-dependent one, HCYS may be metabolized through at least two other pathways not dependent on Cbl.

The measurement of serum metabolite levels has application in the following areas: (1) on account of their sensitivity, as a screening test for diagnosis of Cbl and folate deficiencies; (2) on account of their specificity, to identify true folate and Cbl deficiency among patients with low serum levels of the vitamins; (3) in patients with megaloblastic anaemia and low blood levels of both Cbl and folate, to distinguish Cbl from folate deficiency; and (4) to confirm the diagnosis of Cbl or folate deficiency after treatment and to monitor the adequacy of treatment.

Sensitivity

Current evidence suggests that serum MMA and HCYS assays provide the

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most sensitive tests for detection of Cbl and folate deficiencies. Major contributions to the development and application of serum assays for MMA and HCYS in the diagnosis of Cbl and folate deficiencies have come from the group at the University of Colorado Health Sciences Center working in collaboration with the group from Columbia University College of Physicians and Surgeons in New York (Stabler et al, 1986, 1987, 1988, 1990, 1991, 1993; Allen et al, 1990, 1993a,b,c; Lindenbaum et al, 1988, 1990, 1994; Savage et al, 1994a,b). Using a normal range for these metabolites that spans the log-transformed mean + 3 SD, Stabler et al (1990) found elevated levels of either serum MMA or HCYS in 94% of 86 patients with Cbl deficiency that was defined on the basis of a response to Cbl treatment in patients with a serum Cbl concentration < 200 pg/ml. In a more extensive study by the same group on 406 patients with Cbl deficiency, even better sensitivity was reported using metabolite assays. All but 1 out of 406 patients showed an elevation in either serum MMA or HCYS or both metabolites for a combined sensitivity of 99.8% (Savage et al, 1994a). The sensitivity of the serum MMA was better than the serum HCYS (98.4% versus 95.9%). In the same study, 91% of 123 episodes of folate deficiency in 119 patients were associated with an increase in serum HCYS. Serum MMA was increased in 12.2% of the group with folate deficiency. In all but one of these instances, however, the elevation in MMA was attributed to chronic renal failure or to hypovolaemia. In this study Savage et al (1994a) also noted that MMA assay appeared to be more useful for non-anaemic patients and that HCYS levels were significantly higher in the group with anaemia. In a study carried out on 3846 individuals who had serum Cbl and red cell folate assays, 335 had reduced levels of one or both vitamins (Curtis et al, 1994). After eliminating patients with renal impairment, 72 out of the remaining 281 patients (26%) had elevated levels of serum HCYS. These authors found a poor predictive value for elevated serum HCYS in patients with macrocytosis or low red cell folate (34%). It is not known to what extent the addition of serum MMA, which was not carried out, would have increased the predictive value of metabolite assays in these patients. Also, the specificity of the serum HCYS assay for identifying one or other of the vitamin deficiencies appears to have been good in that 12 out of 13 patients showed a fall in serum HCYS following treatment.

Further evidence to support the sensitivity of metabolite assays to detect early stages of vitamin deficiency come from the studies on patients in early relapse from inadequately treated but previously diagnosed Cbl deficiency (Lindenbaum et al, 1990). These findings are discussed below in reference to the use of MMA and HCYS assays for measuring the efficacy of a therapeutic response to vitamin replacement.

In addition to the observations of raised serum levels of metabolites in patients with clinically significant Cbl and folate deficiencies, there is also evidence of a negative correlation between serum levels of HCYS and either serum Cbl, serum folate or red cell folate in apparently healthy normal volunteers (Israelsson et al, 1988; Andersson et al, 1992; Brattstrtm et al, 1992; Selhub et al, 1993; Jacobsen et al, 1994). This suggests that there


may be a high prevalence of subclinical vitamin deficiency, sufficient to cause an increase in the levels of a serum metabolite, even in persons with normal serum levels of Cbl and folate. On the other hand, Rasmussen et al (1989) found no significant correlation between serum Cbl and MMA levels in 28 healthy volunteers. Joosten et al (1993) measured MMA, HCYS, cystathionine and 2-methylcitric acid in groups of elderly subjects. Elevations in one or more metabolites were found in 63% and 83% of healthy and hospitalized elderly persons, compared with much lower prevalence rates for subnormal serum Cbl, folate and pyridoxine. From this, and based on the assumption that elevated levels of metabolites in the serum represent evidence of incipient vitamin deficiency, these authors concluded that there is a substantially higher prevalence of tissue deficiencies of these vitamins than is apparent from measurement of serum vitamin concentrations. In support of this are the findings reported on seven apparently healthy individuals with normal serum Cbl who had serum MMA elevated above 300 nmol/l. A single injection of cyanocobalamin resulted in a significant fall in their serum MMA levels (Rasmussen et al, 1990a).

It is uncertain whether this degree of metabolic impairment is associated with any morbidity or disease risk, although evidence is mounting that higher plasma levels of HCYS are associated with an increased risk of vascular occlusive disease (reviewed by Kang et al, 1992; Green and Jacobsen, 1995). The causative relationship between lowered vitamin status and raised metabolite levels in otherwise healthy persons is further substanti- ated by reports that vitamin supplementation causes a lowering of those levels. This has been demonstrated for HCYS (Brattstr6m et al, 1988; Ubbink et al, 1993) as well as for MMA (Pennypacker et al, 1992). All these findings point to a need to redefine the normal ranges for serum Cbl and folate levels in the population, yet this would almost certainly result in a greater number of false low results in normal individuals, further com- pounding the problem of poor specificity for serum vitamin assays that has been discussed above. Plasma HCYS level has been used to redefine the lower limit cutoff for plasma folate (Lewis et al, 1992). In a study carried out on 101 patients with coronary artery disease and 108 controls, plasma folate was measured using a microbiological assay and compared with plasma HCYS. Using a defined upper limit for plasma HCYS, these investigators reasoned that plasma folate concentrations inadequate to prevent elevations in plasma HCYS were indicative of biochemical deficiency. On this basis, they defined a 'lower acceptable' plasma folate at 6.6ng/ml (15 nmol/1). Although this approach is an interesting one, these investigators do not appear to have taken into consideration variations in Cbl or pyridoxine status among the subjects.

There is little information on the chronological sequence of changes that occur during developing Cbl or folate deficiencies. It is known that the rate of onset of clinical and laboratory evidence of deficiency is more rapid for folate than for Cbl deficiency. This relates, in large measure, to the relative size of the body stores and the daily requirement for the two vitamins. Consequently, the time from cessation of intake to development of the

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full-blown manifestations of folate deficiency is 4-5 months. This is known from Herbert's meticulous series of observations conducted on himself during a period of dietary folate deprivation (Herbert, 1962). No similar information is available for Cbl, but from observations on the time to development of Cbl deficiency in patients following total gastrectomy, it is known that depletion of normal body stores takes several years (Chanarin, 1979). At what point, during the course of developing deficiency, serum metabolite levels increase above the normal range, is unknown. It is extremely likely that the rise above normal in metabolite levels precedes the fall in serum vitamin levels (Herbert, 1988). This assumption is based, in part, on the description of patients with low normal serum Cbl concentrations who have raised metabolite levels that fall following treatment with Cbl. In some patients, Cbl treatment also resulted in objective improvement in the clinical picture, such as a fall in mean cell volume (MCV: Pennypacker et al, 1992). Additional evidence that the appearance of abnormal serum metabolite concentrations precedes the finding of deficient serum vitamin levels comes from a report of normal serum Cbl (> 200 pg/ml) in 9 out of 173 patients (5.2%) with confirmed Cbl deficiency and raised serum levels of both MMA and HCYS who had no other known explanation for the abnormal metabolites (Lindenbaum et al, 1990). Five of the patients had neurological complaints attributable to the Cbl deficiency and in 11 there were haematological abnormalities.

In the vast majority of patients who have been studied, raised serum metabolites have been found at a single time point which coincides with the finding of lowered serum Cbl or folate levels. It is not ascertainable from this information which occurred first, the raised metabolites or the lowered vitamin levels. Pennypacker et al (1992) noted increased serum metabolite levels in three of their subjects which preceded a fall in serum Cbl by up to 1 year. Additional sequential studies in untreated patients would provide the answer to this question, but are precluded for ethical reasons. Longitudinal population-based studies offer the best alternative, such as the study carried out by Lindenbaum et al (1994) on 548 surviving members of the Framingham Study cohort (Dawber et al, 1951). Of 17 individuals with raised metabolite levels, 13 had serum Cbl levels of 200-350 pg/ml and five of them had either macrocytosis or anaemia. Reports of patients with objective evidence of Cbl deficiency associated with low serum Cbl and normal metabolite levels are rare. Savage et al (1994a) described this situ- ation in only 1 out of a group of 406 patients who were selected on the basis of subnormal serum Cbl levels. Another patient diagnosed as having multiple sclerosis was reported to have pernicious anaemia with low serum Cbl and normal metabolite levels (Ransohoff et al, 1990). The finding of a normal HCYS level in patients with low serum folate and objective evidence of fotate deficiency, however, appears to be somewhat more frequent. In the study reported on 119 patients with folate deficiency by Savage et al (1994a), 11 had normal serum HCYS levels.

Information on the sequence of changes that occur in developing Cbl and folate deficiencies has also been derived from studies on patients previously diagnosed and treated for megaloblastic anaemia caused by Cbl or


folate deficiency. These patients entered haematological relapse because of discontinued or inadequate treatment (Lindenbaum et al, 1990). In all, 42 events were documented in 14 patients and in 13 of those events the serum Cbl was normal (> 200 pg/ml). In six of these events, both the MMA and the HCYS were raised. On the other hand, both metabolites were normal on only two occasions and only on one of these was the serum Cbl level < 200 pg/ml. Elevation of the serum MMA level was the single most frequent abnormal test. These observations are consistent with the concept that changes in serum metabolites that are diagnostic of deficiency precede such changes in the serum Cbl levels.

The clinical usefulness of serum metabolite assays as a sensitive test for diagnosis of Cbl deficiency is most convincingly demonstrated in patients with atypical presentations such as those who display no anaemia or macrocytosis but only neuropsychiatric complications of Cbl deficiency (Lindenbaum et al, 1988; Green, 1994). It is particularly in patients who have neuropsychiatric symptoms that it becomes important to identify or exclude an underlying and potentially correctable Cbl deficiency. In a study of 162 patients admitted on the psychiatry service of a general hospital, six out of ten with low serum Cbl had measurements of serum HCYS and MMA levels carded out and these metabolites were elevated in three, who subsequently proved to have some other objective evidence of Cbl deficiency (Brett and Roberts, 1994). There have been several reports of low serum Cbl values in patients with Alzheimer dementia (Cole and Prchal, 1984; Karnaze and Cannel, 1987; Regland et al, 1988) and in a recent report by Kristensen et al (1993), the Cbl status of patients with Alzheimer dementia was studied using serum MMA levels. These workers found higher mean MlVlA levels in the Atzheimer group than in patients with other dementias, miscellaneous psychiatric disorders affecting the elderly and in age matched healthy controls. Of their 26 Alzheimer patients, 7 (27%) had evidence of Cbl deficiency and in two of these patients, serum Cbl levels were in the low normal range. Macrocytosis was present in only one patient. The authors concluded that Alzheimer patients are particularly prone to Cbl deficiency. It is not known to what extent, if any, the occur- rence and degree of Cbl deficiency among patients with Alzheimer disease contributes to their dementia. It is clearly important, however, to identify and treat the deficiency in the hope of ameliorating at least some compo- nent of the cognitive defects in such patients and in particular, to identify those patients in whom dementia may be due entirely, or in large part to underlying Cbl deficiency (Lindenbaum et al, 1988; Green, 1994). When there is cognitive dysfunction resulting from Cbl deficiency, the opportunity to administer effective treatment depends on the duration of dementia. Early treatment, within 1 year of onset of dementia, is more likely to suc- ceed (Martin et al, 1992).

Lower serum Cbl levels have also been reported in patients with multiple sclerosis compared with age and sex matched controls (Reynolds et al, 1991). In a recent study on 165 patients attending a multiple sclerosis out- patient clinic, 32 (19.4%) had serum Cbl levels < 301 pg/ml. Of these, only seven (4.2%) had elevated MMA or HCYS, leading to the conclusion that

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true Cbl deficiency is not common in multiple sclerosis (Goodldn et al, 1994).

Specificity As discussed above, perhaps as many as one-half of all patients with low serum levels of Cbl do not have demonstrable Cbl deficiency (Stabler et al, 1990; Moelby et al, 1990; Savage et al, 1994a). Once a diagnosis of Cbl deficiency has been made, a patient becomes consigned to lifelong injec- tions of vitamin Bl2. There is therefore a need for a more specific test for Cbl deficiency, to avoid unnecessary long-term treatment in patients who do not have true Cbl deficiency. Serum MMA and HCYS were measured in 196 patients with deficient serum Cbl levels (< 170 pg/ml), and of these 107 (55%) had an elevation in one or both metabolites. In 89 of these patients serum Cbl was < 100 pg/ml, and of this group 82% had abnormal levels of one or both metabolites (Green et al, 1990). In the study reported by Moelby et al (1990), 31 out of 42 patients with low serum Cbl were found to have objective evidence of Cbl deficiency and all but one of these had serum MMA levels higher than 3 SD above the mean for normals. On theoretical grounds, of the two metabolite assays, MMA should be the more specific for diagnosis of Cbl deficiency. Since folate is not involved in the metabolism and conversion of MMA, levels of this metabolite should not be expected to rise in folate deficiency. A raised level of HCYS, on the other hand, does not help to distinguish Cbl from folate deficiency. Measurement of the HCYS level alone may, however, serve as a general indicator of impairment of either Cbl or folate status (Chu and Hall, 1988; Hall and Chu, 1990). In 123 incidents of folate deficiency, 15 (12.2%) were associated with raised serum MMA but in all except one, the raised MMA could be attributed to coexistent renal failure or plasma volume contraction (Savage et al, 1994a). Because renal failure can result in accumulation of MMA (Rasmussen et al, 1990b), the specificity of serum MMA measurement for detection of Cbl deficiency is reduced in the presence of renal disease. It does not appear that heterozygotes for inherited forms of methylmalonic acidemia show increased MMA levels. In six obligate heterozygotes for this disorder, serum and urine MMA levels were normal (Rasmussen and Nathan, 1990).

As discussed above there are serious shortcomings in specificity for the diagnosis of folate deficiency using currently configured tests for direct measurement of the vitamin in the blood and this applies particularly to the red cell folate assay (Chanarin, 1979; Lindenbaum and Allen, 1995). By itself, serum HCYS measurement has poor specificity, because levels of this metabolite rise both in Cbl and folate deficiencies as well as several other acquired and inherited conditions (Green and Jacobsen, 1995). However, in the clinical setting of a patient with suspected Cbl or folate deficiency, measurement of serum HCYS is useful if carded out in conjunction with serum MMA determination. Savage et al (1994a) found an increased level of serum HCYS with normal MMA in 85.7% of patients with folate deficiency but in only 1.3% of patients with Cbl deficiency.


They calculated that the specificity for folate deficiency of the finding of an isolated elevation of HCYS was 98.7%.

Distinguishing Cbl from folate deficiency

To distinguish Cbl or folate deficiency, it is helpful to measure serum levels of both vitamins in addition to metabolite levels. As discussed above, serum assays for Cbl and folate, on their own, may not provide sufficient information by which to distinguish deficiencies of the two vitamins. In conjunction with the metabolite assays, however, they can enhance overall diagnostic efficiency (Allen, 1992). Of the two metabolites, MMA is more useful than HCYS for distinguishing Cbl from folate deficiency, since raised levels of MMA have rarely been described in folate deficiency not associated with any other known cause of methylmalonic acidaemia (Stabler et al, 1990). Measurement of HCYS alone cannot distinguish folate from Cbl deficiency (Chu and Hall, 1988; Hall and Chu, 1990).

There have been several attempts to use other metabolites to help in the differential diagnosis of Cbl and folate deficiency. Allen et al (1993c) have shown that patients with Cbl deficiency have raised serum levels of the two isomers of 2-methylcitric acid in addition to raised MMA and they have raised serum levels of cystathionine in addition to HCYS. Both the frequency and the degree of rise in 2-methylcitric acid are lower than for MMA, so that the measurement of this metabolite does not offer any advantage over measurement of serum MMA for diagnosis of CbI deficiency (Allen et al, 1993b). It is curious that levels of 2-methylcitric acid are below normal in 40% of patients with folate deficiency (Allen et al, 1993c), since MMA is not similarly affected. Again, however, measurement of 2-methytcitrate is not likely to provide any diagnostically useful information, since in Cbl deficiency, the MMA level is almost invariably raised, which provides sufficient information to make a distinction from folate deficiency.

In patients with folate deficiency, levels of cysthathionine, N,N- dimethylglycine and N-methylglycine are raised in addition to serum HCYS (Allen et al, 1993b). Measurement of cystathionine offers little if any added specificity for distinguishing folate deficiency from Cbl deficiency. Of 30 patients with Cbl deficiency, 26 (87%) had elevated cystathionine levels compared with 19 out of 20 (95%) patients with folate deficiency (Stabler et al, 1993). Serum cystathionine does, however, help to eliminate CBS deficiency in the differential diagnosis of an elevated serum HCYS level, since in this inherited disorder, serum cystathionine levels are low or low normal (Stabler et al, 1993). On the other hand, measurement of serum N,N-dimethylglycine and N-methylglycine levels may be useful in distinguishing Cbl from folate deficiency. Of 25 patients with folate deficiency, 19 (76%) and 15 (60%) showed increases in the levels of N,N- dimethylglycine and N-methylglycine levels respectively. Only 9 out of 50 patients (18%) with Cbl deficiency showed such increases. These preva- lences were based on a cutoff defined by the mean + 2 SD for the normal range (Allen et al, 1993b). It is perhaps noteworthy that no patients with

556 R. GREEN

Cbl deficiency had serum N-methylglycine levels above the normal range. These findings suggest a possible clinical utility for measuring these other metabolites in patients with raised serum HCYS levels in whom Cbl deficiency cannot otherwise be excluded.

Efficacy of therapeutic response

Measurement of serum metabolite levels is not only useful for establishing a diagnosis of Cbl or folate deficiency and for distinguishing between these two deficiencies, but also, to confirm that raised levels of one or both of these metabolites responds to treatment with the particular vitamin that is considered to be deficient. Follow-up measurement of an abnormal serum metabolite level also provides the opportunity to monitor efficacy of treatment. Thus, Lindenbaum et al (1988) showed a fall in the serum levels of MMA and HCYS in all of 31 patients with Cbl deficiency 1-5 days after treatment with cyanocobalamin. Just as a return to normal of serum metabolite levels may be taken as evidence of a response to therapy, persistence of elevated levels are indicative of a failure of response or relapse. The same group also studied 44 patients previously diagnosed with Cbl deficiency during 243 follow-up visits (Lindenbaum et al, 1990). In 14 of these patients, seen on 42 occasions, mild haematological relapse was identified based on the presence of hypersegmented neutrophils in the blood smear, or an increase in the MCV of 5 fl or more. On 13 occasions (31%), the serum Cbl level was > 200 pg/ml. Metabolite assays, however, provided a more sensitive laboratory indication of relapse, with 41 patients showing an elevation in one or both metabolites (39 had raised serum MMA). In another report, all of 20 patients who responded to treatment with cyanocobalamin for confirmed Cbl deficiency showed a marked fall in serum MMA, and in 18 there was a return to normal (Moelby et al, 1990).

The usefulness of serum metabolite assays to confirm the diagnosis by assessment of the therapeutic response to administration of a specific vitamin is demonstrated in the report by Allen et al (1990). Five patients with Cbl deficiency showed no fall in serum MMA or HCYS in response to treatment with folic acid. All showed a response following Cbl treatment. In a further three patients with folate deficiency, treatment with cyanocobalamin did not cause lowering of serum HCYS, whereas folic acid treatment did. In a patient with isolated elevation in serum HCYS, it would be reasonable to assume that there is an underlying deficiency of folate, and to treat with this vitamin. However, Allen et al (1990) report that in approximately 10% of patients with low serum levels of both folate and Cbl, serum HCYS alone may be elevated (usually both metabolite levels are abnormal). Under these circumstances, and because low serum Cbl may occur in almost 50% of patients with folate deficiency (Mollin et al, 1962), it would be reasonable to carry out a therapeutic trial with folic acid alone, and to then monitor the HCYS level for a response. Following treatment with the appropriate vitamin, it may take up to 1 week for serum MMA and HCYS levels to return to normal (Lindenbaum et al, 1988; Stabler et al,


1990). The persistence of elevated metabolite levels for several days following treatment may be useful in patients who were Cbl or folate-deficient but then received either of these vitamins. Serum levels of Cbl or folate will rise immediately following treatment (or following blood transfusion in the case of folate), thus obscuring the underlying cause of deficiency. During the first several days after such treatment, raised levels of metabolites may still be diagnostic. Furthermore, because of the time lag in response of serum metabolite levels, follow-up measurement of MMA and HCYS should be measured 7-14 days after treatment in order to assess the efficacy of response.

Other causes of raised serum metabolites

Renal failure results in an increase in serum levels of MMA and HCYS. The rise in HCYS levels has been reported extensively in the literature (Wilcken et al, 1981; Kang et al, 1983; Sofia et al, 1990; Chauveau et al, 1992; Hultberg et al, 1993a). A rise in serum MMA levels has also been reported in renal disease. Moelby et al (1992b) reported increased serum MMA in 36 out of 37 patients with end stage renal disease. Coexistent Cbl deficiency was ruled out by normal serum Cbl levels and by failure to nor- malize serum MMA following injection of cyanocobalamin. The observations that serum MMA and HCYS are both elevated in patients with renal failure limits the usefulness of these assays for diagnosis of Cbl and folate deficiency in patients with coexistent renal disease. Norman and Morrison (1993) have pointed out that this limitation does not apply to the measurement of urine MMA. It has been reported that patients with hypothyroidism who have no evidence of Cbl or folate deficiency may have raised levels of serum HCYS (Chong et al, 1993; Green and Jacobsen, 1995). In addition to these acquired disorders there are several inherited enzyme defects that can cause an increase in one or both of the metabolites. Individuals who are homozygous for these defects are usually first seen during infancy or childhood and often have markedly elevated levels of the affected metabolites. As discussed above, elevated levels of s e r u m

HCYS occur in abnormalities affecting both the transsulphuration and the remethylation pathways for HCYS metabolism (Mudd et al, 1995; Fenton and Rosenberg, 1995b). Among the methylation defects are several affecting Cbl processing enzymes, including some which can also cause methylmalonic acidaemia. Other defects in Cbl processing enzymes cause only serum MMA level to rise (Fenton and Rosenberg, 1995b). Heterozygotes show varying phenotypic expression in the degree of elevation of serum metabolite levels, depending on the nature of the defect, the integrity of alternative pathways (in the case of HCYS) and possibly the nutritional status of B-group vitamins involved in the pathways. For example, individuals heterozygous for CBS or MeTHFR defects frequently have mild to moderate elevations in serum HCYS (Boers et al, 1985; Clarke et al, 1991; Rosenblatt, 1995). On the other hand, obligate heterozygotes for methylmalonic acidaemia do not have raised serum MMA levels (Rasmussen and Nathan, 1990).

558 R. GREEN

Do metabolite patterns offer clues to the pathogenesis of disease in Cbl and folate deficiency?

The complications that result from Cbl and folate deficiency may result from inadequate production of an essential product of Cbl or folate-dependent reactions. Alternatively, complications may arise as a result of the harmful effects of substrates that accumulate in excess during the vitamin deficiencies. Thus, methylmalonate has been implicated in the defective myelin synthesis that occurs in Cbl deficiency (Green and Jacobsen, 1990; Metz, 1992) and it has been suggested that HCYS may have neurotoxic effects. The possible role of MMA and other metabolites such as 2-methylcitrate that accumulate in the serum and CSF in Cbl deficiency in the pathogenesis of nervous system damage has also been considered (Stabler et al, 1991; Allen et al, 1993b). Thus far, however, no consistent pattern of these metabolites has been described in patients with, compared to those without, neurological complications of Cbl deficiency (Allen et al, 1993b). It has also been proposed that chronic, long-standing elevations in HCYS level, attributable to mild degrees of folate or Cbl deficiency, may be responsible for degenerative cardiovascular changes including atherosclerosis and thrombosis. There is considerable evidence to support this view and the topic has been reviewed extensively elsewhere (Ueland and Refsum, 1989; Kang et al, 1992; Green and Jacobsen, 1995).

SUMMARY

Cbl and folate are both necessary for the metabolism of HCYS, whereas only Cbl is required for MMA metabolism. During the past decade, analytical methods have been developed that are sensitive enough to detect low levels of MMA and HCYS normally present in the plasma. These methods are sufficiently precise to be used in the clinical laboratory and measurements of the serum levels of the metabolites provide sensitive and specific techniques for the identification of Cbl and folate deficiencies. These techniques constitute an important addition to the battery of diagnostic tests that are available for detecting the vitamin deficiencies and for distinguishing each from the other. By virtue of the role of Cbl and folate in the metabolic pathways that involve MMA and HCYS, levels of both metabolites rise in Cbl deficiency, but only HCYS rises in folate deficiency. During the development of Cbl or folate deficiencies, accumulation of these metabolites in the plasma signals the existence of a condition of biochemical vitamin deficiency of sufficient degree to cause impairment in the metabolic pathways which are dependent on these vitamins. Circulating metabolite levels appear to accurately reflect the nutritional status of the vitamins and a rise in serum metabolite levels is therefore one of the earliest and most reliable indicators of developing Cbl and folate deficiencies. Elevations of serum metabolites above the reference range not only precede a fall in the serum vitamin levels but also show a more consistent correlation with objective evidence of vitamin deficiency than do low blood vitamin levels.


The advent of serum metabolite measurements has also made it possible to identify subtle or atypical forms of vitamin deficiency that may be associated with unusual or previously undiscovered disease manifestations. Thus, in patients who display only neurological manifestations of disease, underlying Cbl deficiency may be revealed by the finding of raised serum or urine levels of MMA. Similarly, unsuspected folate deficiency may be disclosed by the finding of a raised serum HCYS. This may have important implications with respect to disease risk, since there is mounting evidence that sub-optimal folate nutritional status may be associated with increased risks of vascular disease, neoplasia and birth defects. Finally, the measurement of serum levels of MMA, HCYS and other metabolites that accumulate in Cbl and folate deficiencies may provide important new insights into the mechanism whereby these vitamin deficiencies lead to different patterns and manifestations of disease.

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6 metabolite assays in cobalamin and folate deficiency

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