MA1 MA2 MA3 MA4 MA5 P-REF LMP
4.1.2.3 Demanda bioquímica de oxígeno (DBO 5 )
Introduction
Bacterial colonisation o f the small bowel may lead to anomalies in both vitamin B, 2
absorption (Rosenberg and Mahoney 1973) and propionic acid metabolism. Urinary concentrations o f methylmalonic acid are seen to increase with inadequate intestinal absorption of vitamin B^^ or the increased production o f propionic acid by intestinal bacteria, whilst compromised vitamin B,2metabolism may occur as a consequence o f
either the compromised activity o f the essential cofactor 5' adenosyl cobalamin. In the treatment o f methylmalonic aciduria, the administration o f oral antibiotics such as Neomycin (Snyderman et al 1972) has been shown to reduce urinary excretion of
methylmalonic acid, suggesting that in the event o f bacterial infection of the small intestine, bacteria may significantly contribute to the methylmalonate pool.
The aim o f this section of the thesis was to determine whether the quantitation o f urinary methylmalonic acid in patients with failure to thrive and/or diarrhoea was a viable indicant of small bowel bacterial colonisation. Samples from patients with known colonisation were compared with those o f patients with other known gastrointestinal anomalies. A control group o f children with no known gastrointestinal disorders was also investigated to
determine the specificity of the method of analysis used. A new rapid and sensitive GC-MS method was established to facilitate the investigation o f this hypothesis.
9.1.1 Methylmalonate Metabolism.
Methylmalonate is a conjugate base, that, in the form of Coenzyme A esters, plays an important role in intermediary metabolism. In normal circumstances trace concentrations of methylmalonic acid are found in urine, blood and cerebrospinal fluid. Methylmalonic acid is an intermediate in conversion o f propionate to succinyl CoA prior to the latter compounds entry into the tricarboxylic acid cycle. As shown in Fig 9.1 propionyl CoA acid is first converted to d-methylmalonyl CoA by the reaction o f propionate carboxylase (with biotin as a co-factor). Racemation facilitated by the enzyme methylmalonyl racemase leads to the production o f 1-methylmalonyl CoA which in turn is converted to succinyl CoA by methylmalonyl CoA mutase. For this final reaction the vitamin B, 2 cofactor 5'
deoxyadenosyl cobalamin is required (Thompson et al 1989). Vitamin B^2is not
ISOLEUCINE, M ETHIONINE THREONINE, CHOLESTEROL
ODD CHAIN FATTY ACIDS
1
SO On
GUT BACTERIA _____ ^ PROPIONATE _____ ^ PROPIONYL CoA
ATP,Mg**.biotin
1
propionyl CoA carboxylaseD METHYLMALONYL CoA
THYMINE, VALINE --- ► .
* methylmalonyl CoA racemase
L METHYLMALONYL CoA
Figure 9.2 shows the production of 5' deoxycobalamin. Following absorption vitamin B,2,
now in the form of a hydroxycobalamin, is released from the complex and transported to body tissues by the and serum globulins, transcobalamin I and II. Transcobalamin I (TCI) is associated with the transport o f plasma hydroxycobalamin and its storage in the liver, whilst transcobalamin II (TCII) is responsible for the tissue transportation of hydroxycobalamin (Rosenberg and Mahoney 1973). TCII enters cells bound to hydroxycobalamin by means of a calcium ion dependent receptor mediated process (Willard and Rosenberg 1980).
Hydroxycobalamin (B^^a) Co(III)
i
Hydroxycobalamin (B,jb) Co(II)i
Hydroxycobalamin (B.^c) Co(I)i
5' deoxyadenosyl cobalaminL Methylmalonic CoA --- ► Succinyl CoA
Fig 9.2 Production of 5' Deoxycobalamin.
Absorbed hydroxycobalamin has a central cobalt unit [Co(III)] which is reduced by two reductase enzyme reactions to Co(II) and Co(I). 5' deoxyadenosyl cobalamin is then formed by an energy dependent transferase reaction .
The incidence o f Methylmalonic Aciduria can be attributed to three main events; Disordered cobalamin metabolism, vitamin B, 2 deficiency or propionic acid metabolism
disorders. Anomalies in propionic and vitamin Bjj metabolism leading to increased production o f urinary methylmalonic acid also occur as a result o f various gastrointestinal disorders. Deal absorption of vitamin Bjj is significantly reduced in the incidence o f small intestinal mucosal damage when the specific receptors for the binding of the intrinsic factor-B, 2 complex are absent or reduced. This in turn leads to a deficiency in the cofactor
5' deoxyadenosyl cobalamin and subsequent accumulation of methylmalonic acid.
Similarly in small intestinal bacterial infestation, available Vitamin Bj2 may be utilised by
colonising organisms for the production o f bacterial glutamic acid and ribonucleotide reductase (Brock 1984). An additional source of urinary methylmalonic acid are propionic acid producing bacteria present in the gut (Barshop et al 1991). Thus the accurate trace analysis o f urinary methylmalonic acid may be an important index of small intestinal bacterial overgrowth.
9.1.2 Isolation and Analysis o f Methylmalonic Acid.
The isolation o f methylmalonic acid from biological fluids is an important procedure for the early detection o f the congenital organic disorders that manifest as methylmalonic aciduria. Various methods have been devised for the isolation and detection of
methylmalonic acid. Early colourimetric and paper chromatography methods (Cox and White 1962, Giorgio and Plaut 1965, Oberholzer et al 1967) were subject to lack of sensitivity and specificity and required lengthy preparation prior to analysis which led to unaccountable losses of the analyte of interest. Samples were first either acidified to facilitate isolation as a function of their polarity (Cox and White 1962) or subjected to overnight ether extraction and then separated by ion exchange chromatography prior to final colourimetric analysis. Use o f gas chromatography procedures (Mamer and Tjoa
9.2 Method Optimisation.
As both methylmalonic and oxalate are organic acids, it was initially thought that the method used for the isolation and detection of urine oxalate could be extended to incorporate methylmalonic acid, using oxalate as a combined internal standard. However when this was investigated initial results were unsatisfactory, due mainly to the
1 0 fold difference between the normal urine concentrations of oxalate and methylmalonate.
This made the construction of a valid combined calibration curve extremely difficult, (see 3.5). For this reason the analysis of urine methylmalonic was performed separately and a more suitable internal standard was sought. Malonic acid was eventually chosen as it adhered to internal standard criteria (see 3.5).
Various concentrations of malonic acid were chosen to find the concentration that would provide the most suitable peak shape and intensity for the standard methylmalonic acid concentrations. Fig 9.3 shows the structures of methylmalonic and malonic acid.
Methylmalonic Acid Malonic Acid
COOH COOH
HoC— '3
i
COOH COOH
(mol wt 118.09) (mol wt 104.06)
Fig. 9.3 Structures of analysed compounds.
When derivatised with t-BDMS, the malonic acid fragment, 275.11 m/z (M^-15), was found to have an ionic intensity (mV) approximately ten times greater than that o f the silyated methylmalonate fragment, 289.13 m/z (M^-15). Thus the final 2:1 ratio o f malonic acid to methylmalonic acid was attained with malonic acid at a concentration o f 5mmol/1.