New diet conceptsin Propionic acidaemia andMethyl malonic acidaemia

Human gut microbiota

▪ complex microbial community consisting of 100 trillion microbes in the intestine

▪ gut microbiota is essential to the health and well-being of the host

▪ gut microbiota fluctuates in response to nutritional uptake rather than remaining stable

▪ diet and medication is a key modulator in gut microbiota composition

GeographyBirth mode Breast feeding

Diet

Drugs

Exercise

Disease Ageing

Gut microbiota

Dietary intake, especially of

nondigestible carbohydrates, alters

the species composition of the gut microbiota both short term and

long term

Dietary intake, especially of

nondigestible carbohydrates, alters

the species composition of the gut microbiota both short term and

long term…. leads to production of

short chain fatty acids:

acetic acidbutyric acid

propionic acid

Dietary intake, especially of

nondigestible carbohydrates, alters

the species composition of the gut microbiota both short term and

long term…. leads to

production of short chain fatty

acids:acetic acid

butyric acidpropionic acid

Propionate mainly produces glucose in

the liver.Acetate and butyrate are incorporated into other fatty acids and

cholesterol

Probiotic intake

Dietary fibre intake

Altered pH

Protein intake

Sugar intake

Fat intake

Antibiotics

↓ SCFA production

↑ lipopolysaccharide production

↑TMAO production

Gut inflammation

Cognitive decline

↑ risk of diarrhoea

Insulin resistance

↑ CVD risk ↓ risk of infections

Insulin sensitivity

↓ gut inflammation

Improved lipid metabolism

↓ kidney toxins

↑ antioxidant production

↑SCFA production

Disease Health

Main principles of MMA/PA therapy

▪ prevent recurrent episodes of ketoacidosis and hyperammonaemia

▪ reduce accumulation of toxic tissue metabolites (organic acid compounds)

▪ support anabolism, normal growth and nutritional status

Objectives of nutritional treatment

▪ substrate restriction: isoleucine, methionine, threonine and valine by natural protein restriction

▪ +/-MMA/PA-free amino acids

▪ maintain adequate energy intake

▪ limit fasting time to oxidation of odd chain fatty acids

▪ micronutrient/LCP supplementation

▪ maintain adequate hydration

▪ prevent catabolism during illness

Sources of propionate in MMA and PA

Propiogenic precursors

Amount

Diet and muscle turnover of propiogenic amino acids: methionine, threonine, valine, isoleucine

approx 50%

Oxidation of odd-numbered long chain fatty acids

approx 25%

Gut microbiota approx 25%

Cholesterol small

Propionate production

▪ Biochemical pathways (acrylate, propanediol, and succinate) contribute to propionate formation by gut bacteria

▪ Propanediol pathway is an important source of gut propionate▪ Present in Lachnospiraceae related to Ruminococcus spp. and Roseburia inulinivorans

Fucose and rhamnose are metabolized by the propanediol pathway

Gut microbiota in MMA/PA patients

Lack of information about the composition and or/functioning of the gut microbiota in MMA/PA patients

Breast feeding

Antibiotics Carnitine Constipation

Gut microbiota

Low fibre intake

Tube feedingLow fluidintake

Protein amountand source

Impact of antibiotics

▪ aim: ↓ intestinal microbiota responsible for propionate production

▪ appear to ↓ levels of propionate in MMA/PA urine and plasma

▪ limited ‘supporting’ scientific data

▪ MMA/PA guidelines (2014) recommend metronidazole as an option to ↓ intestinal microbiota responsible for propionate production

Impact of antibiotics

▪….but decrease bacterial population and diminish species diversity

▪ affecting balance of normal gut microbiota

odrug resistance

o toxicity/safety concerns

ometronidazole not effective in reducing faecal excretion of SCFA production (Hoverstad et al 1986)

L- carnitine

▪ L-carnitine compensates for secondary carnitine deficiency

▪ not absorbed until reaches the large intestine

▪ uptake of carnitine by the colon influenced by the gut microbial population

▪ low fibre intake can stimulate carnitine breakdown by enteric microbiota

▪ changed to trimethylamine (TMA)

▪ further metabolised to trimethylamine N-oxide (TMAO)

▪ TMAO has been linked to atherosclerosis

Constipation common in MMA/PA

▪ constipation contributes to metabolic instability

▪ constipation appears to precede decompensation

▪ ↑ time of bacterial contact with the stool in the gut

▪ ↓ fibre intake

▪ inadequate fluid

▪ ↓ gut motility

▪ ↓ physical activity /developmental delay

▪ muscle hypotonia

Laxative therapy

▪ Patients (n =4) with PA given Senokot /gut motility agents:

- ↓ ammonia

- ↓ urinary excretion of propionyl glycine

- ↑ free and total carnitine

- suggestion: less propionate generated by shortening the time of bacterial contact with stool in the gut

Prasad C, Nurko S, Borovoy J, et al. J Pediatr 2004;144:532-5

Why low fibre intake in MMA/PA?

▪ low protein diet

▪ lack of cereal fibre

▪ lack of appetite

▪ nausea

▪ vomiting

▪ tube feeds

olow fibre intake =↓ production of short chain fatty acids

Fibre intake in MMA/PA

3 m 6 m 9 m 1 2 m1 5 m1 8 m 2 y 3 y 4 y 5 y 6 y 7 y 8 y 9 y 1 0 y 1 1 y 1 2 y 1 3 y 1 4 y 1 5 y

0

5

1 0

1 5

2 0

A g e (m o n th s / y e a rs )

Fib

re

in

tak

e (

g/d

ay

)

n=14

Suggested BNF /RNI for fibre from age 5y =18g/day

Tube feeding

▪ amino acids (elemental diets) lack dietary fibre so may suppress colonic fermentation

▪ natural protein source: may be reluctance to use protein source with added fibre/prebiotics

3 m 6 m 9 m 1 2 m1 5 m1 8 m 2 y 3 y 4 y 5 y 6 y 7 y 8 y 9 y 1 0 y 1 1 y 1 2 y 1 4 y 1 5 y 1 6 y

0

1

2

3

A g e (m o n th /y e a r )

To

tal

pro

tein

in

tak

e (

g/k

g/d

)

fro

m n

atu

ra

l a

nd

pre

cu

so

r-f

re

e L

-am

ino

ac

ids

n a tu ra l p ro te in g /k g /d a y

p re c u s o r - fre e L a m in o

a c id s g /k g /d a y

s a fe le v e l o f p ro te in in ta k e

W H O /F A O /U N U 2 0 0 7

13y

n =1 0

n = 1 3

n =1 2

n =1 2n =1 3 n =1 2n = 8

n =1 0

n = 9

n = 3

n = 1n = 1

n = 7n = 4

n = 2

n = 4 n = 4n = 3

n = 4

n = 4

n = 1

p = 0 .0 3

Protein intake of MMA/PA children

Nutrini Multifibre

▪ 50% soluble, 50% insoluble fibre

▪ soy polysaccharides

▪ resistant starch

▪ inulin

▪ arabic gum

▪ cellulose

▪ oligo fructose

Natural protein source with Multifibre

1 2 3 4 5 6 7 8 9 10 11 120

20

40

60

80

100

% in

take

Fluid intake: median 67% of recommended fluid for age n=12

Fluid intake of children on tube feeds

▪when giving tube feeds only, it is difficult to maintain adequate fluid intake

Gut microflora

Human milk oligosaccharides

❖Bifidobacteria❖Lactobacilli

Breast fed infant

Formula fed infants

❖Enterobacteriaceae❖Enterococci❖Bacteroides❖Clostridia❖Bifidobacteria

Bifidobacterium sp. ferment dietary oligosaccharides = SCFAs production

Lower breast feeding

▪ From birth, MMA/PA microbiota likely to be different

▪ Low rate of breast feeding in MMA/PA

▪ At best, expressed breast milk provides part of requirements

▪ glucose liberated from polymers in emergency diets

▪ theoretically may increase production of propionate in the gut

Emergency feeds based on glucose polymer

Trial background

Necessary to build knowledge as an important first step to find alternative ways to reduce propionate production and constipation in PA

▪ no evidence about gut microbiota composition in PA patients

▪ dietary fibres required to alleviate constipation

▪ known to influence the composition and activity of gut microbiota and,

consequently, the production of SCFA

▪ no evidence on an optimal fibre composition in PA patients

Project aims

1. What is the phylogenetic composition of the gut

microbiota of PA patients?

a.How large are the inter-individual differences

between PA patients?

b.How large are the intra-individual difference in PA

patients in a time span of 3 months?

2. Which fibres, or fibre combinations, reduce propionate

production in faecal slurries from PA patients in a batch

fermentation system?

Design and sampling timeline

PA patients, Age >3 yr and preferably <10 yr (upper age limit is 35 yr)

Inclusion:Having PA (or having a sibling with PA) and age >3 yr and preferably <10 yr (upper age limit is 35 yr).Exclusion:PA patients with confounding intestinal diseases (e.g. colon rectal cancers, Crohn's disease, Ulcerative colitis).

Planned characterization assays

▪ Physiological parameters

(PH, SCFA, Lactate, Secretory IGA)

▪Microbiota community profiling

(Next Generation Sequencing technology ‘MiSeq’ )

▪ Fibre fermentations on PA patient samples

Additional exploratory analysis

(Gene qPCR, Proteomics, Metabolomics)

MF6/ fibre blend

Oligofructose

Arabic gumInulin

Resistant starchSoy polysaccharides

Cellulose

▪ Fibre blend provides the range and type of fibres habitually consumed in the normal diet

▪ Customized MF6 + Guar gum: ~71% soluble and ~ 29% insoluble fibres

Amino acid supplements with fibre blend for MMA/PA

Study question and methods

McFall-Ngai et al. 2013 PNAS

What is the effect of fibre mixture on propionic acid production?

In vitro fermentation system: i-screen

Results: propionate profile

Fibres tested:

phGGA: Optifiber

phGGB: Benefiber

6 fibre mix (No GG): Nutrini MF6 mix 8

6 fibre mix (GG):Anamix MF6

▪ Fibre mixture in Anamix Junior has lower propionate production potential in this

fermentation system as compared to other tested fibre blends

Microbiota composition showed minordifferences between the fibre mixtures

Principal Coordinate Analysis, based on operational taxonomic unit abundance, of microbiota beta-diversity as measured in weighted UniFrac distances

MF6-AnamixMF6-mix 8BenefibreOptifibre

▪ Microbiota composition is comparable between fibre blends

Discussion

▪ propionate, precursor of propionyl-CoA, is one of the main short chain fatty acids produced by gut microbiota

▪ several aspects of treatment may ‘negatively’ impact on gut microbiota

▪ no evidence about gut microbiota composition in PA/MMA patients

▪ studies now looking at microbiota composition and impact of different fibres on propionate production

Discussion