Úspešnosť diéty s obmedzením prísunu bielkovín pri pacientoch trpiacich alkaptonúriou je závislá od veku
V. DE HAAS1, E. C. CARBASIUS WEBER1, J. B. C. DE KLERK2, H. D. BAKKER3,
G. P. A. SMIT4, W. A. R. HUIJBERS5, M. DURAN1, and B. T. POLL-THE1*
1 University ChildrenÏs Hospital Het W ilhelmina KinderziekenhuisÏ Utrecht;
2 Sophia ChildrenÏs Hospital, Rotterdam; 3 Emma ChildrenÏs Hospital, Academic
Medical Center, Amsterdam; 4 Beatrix ChildrenÏs Hospital, Groningen; 5 Beatrix
Hospital, Gorichem, T he Netherlands
* Correspondence: University ChildrenÏs Hospital, Het W ilhelmina
KinderziekenhuisÏ, Nieuwegracht 137, 3512 L K Utrecht, T he Netherlands
MS received 17.3.98 Accepted 29.5.98
Zhrnutie: Alkaptonuria is characterized by an increased urinary excretion of
homogentisic acid, pigmentation of cartilage and connective tissues, and ultimately
the development of inÑammatory arthropathy. Various diets low in
protein have been designed to decrease homogentisic acid excretion and to
prevent the ochronotic pigmentation and arthritic lesions. However, limited
information is available on the long-term beneÐcial eects of these diets. We
reviewed the medical records of 16 patients aged 3È27 years (4[18 years) to
ascertain the age of diagnosis, growth, development, social behaviour, signs of
complications and longitudinal dietary compliance. The diagnosis of alkaptonuria
was made at an average age of 1.4 years (2 monthsÈ4 years) ; following the
diagnosis all patients were prescribed a diet with a protein content of 1.5 g/kg
per day. All patients showed normal growth and development, and no major
complications of the disease. Behavioural problems associated with poor
dietary compliance emerged as the main problem. Dietary compliance
decreased progressively with age. The eect of dietary protein restriction in
homogentisic acid excretion was studied by Ðxing the amounts of protein in the
diet at 1 g/kg per day and 3.5È5 g/kg per day during 8 days. Twelve patients,
aged 4È27 years, participated in the investigation. Protein restriction resulted in
a signiÐcantly lower excretion of homogentisic acid in the urine of children
younger than 12 years (p\0.01), whereas this eect was less obvious for adolescent
and adult patients. The results suggest that restriction of protein intake
may have a beneÐcial eect on alkaptonuric children ; but continuation of this
regimen to older age seems questionable and not practical.
Alkaptonuria is an inborn error of metabolism (MuKusick 203500) caused by a
deÐciency of homogentisate-1,2-deoxygenase (HGO, EC 188.8.131.52), present in (at
792 de Haas et al
least) the liver and the kidney. This enzyme deÐciency results in the accumulation of
homogentisic acid (HGA), an intermediary metabolite in the catabolism of the
amino acids phenylalanine and tyrosine (La Du et al 1958; OÏBrien et al 1963). A
major part of HGA is excreted into the urine and imparts a characteristic blackening
of urine upon oxidation. The polymer derived from HGA, a melanin-like
product, is deposited in connective tissue and cartilage, causing a pathological pigmentation,
known as ochronosis (Gaines 1989). However, the mechanism by which
the biochemical defect leads to the development of ochronosis remains to be identi-
Ðed. Over the years, the main complications of the disease are ochronotic arthropathy, cardiovascular ochronosis, urogenital obstruction due to ochronotic calculi,
and ocular and cutaneous ochronosis. The clinical complications of this ochronosis
usually become evident by the fourth decade and tend to aggravate with age
(Bosacco et al 1991; Gaines 1989; Konttinen et al 1989; Martin and Batko 1987).
Attempts to treat alkaptonuria have been directed mainly towards prevention of
ochronosis. Although dietary protein restriction has been proposed as a means of
treatment by decreasing the output of HGA, quantitative results and longitudinal
data are scarce. Several groups have studied the efficacy of treatment with ascorbic
acid. In rats with induced experimental alkaptonuria, ascorbic acid reduced the
binding of homogentisic acid to connective tissues (Lustberg et al 1970). It has
therefore been suggested that megadoses of ascorbic acid may prevent the development
of ochronosis in alkaptonuria patients. Unfortunately, this eect is difficult to
measure and deÐnite proof of efficacy will require a long-term study (Sealock et al
1940; Wol et al 1989). In contrast, the alkaptonuric mouse that is homologous to
human alkaptonuria does not show any signs of ochronosis or arthritis
To gain longitudinal information on the clinical, neurological and development
status of children with alkaptonuria, hospital records of 16 patients were studied
along with a detailed clinical investigation. In 12 patients we investigated the eect
of dietary protein restriction on HGA excretion by analysing the excretion of HGA
after a protein intake of 1 g/kg per day versus the excretion after a protein intake of
5 g/kg per day. The eects of these dietary manipulations were compared with those
of the usualÏ diet of the patients.
SUBJECTS AND METHODS
Patients : Sixteen alkaptonuric patients (14 males, 2 females), aged 3È27 years, were
studied. Four patients were older than 18 years. All were diagnosed on the basis of
having dark urine and urinary HGA excretion. Clinical and biochemical data were
ascertained from medical records, to assess the neonatal period, diagnosis, development,
clinical signs of complications, social behaviour, longitudinal dietary compliance
and HGA excretion. No formal psychological testing was performed. Informed
consent was obtained from patients older than 18 years and from the parents of
each patient aged less than 18 years.
Diets : All subjects were studied when clinically well. Their usual dietary habits
were recorded as Diet 0. Twelve patients agreed to participate in a study with two
J. Inher. Metab. Dis. 21 (1998)
Dietary protein restriction in alkaptonuria 793
dierent diets and underwent a two-study protocol, each of which lasted for 8 days.
For each protocol, the patient was provided with a standardized dietary prescription.
The Ðrst diet (Diet 1) consisted of a protein intake of 1 g/kg per day for 1 week.
The second diet (Diet 2), which followed immediately after the Ðrst week, consisted
of an intake to provide 5 g/kg per day of protein for 1 week. It is important to
realize that on Diet 0, the average intake of protein in children was 1.1È1.2 g/kg per
day. Although Diet 0 was designed to provide about 1.5 g/kg per day of protein,
most parents were anxious to keep strictly to the diet, resulting in a true protein
intake which was lower than prescribed.
For the adolescents and adults, it was very difficult to reach the high protein
intake of 5 g/kg per day prescribed in Diet 2. Therefore, the highest possible protein
intake was approximately 3.5 g/kg per day. We are convinced that this lower intake
was not a consequence of poor compliance. All children were seen frequently by
their treating physician during the two weeks of the study and the dietary intake
was controlled by their highly motivated parents. As 5 g/kg per day is an enormous
protein intake for the older group of patients, the amount of 3.5 g/kg per day is the
most reasonable intake that could be accomplished. This diet was also prescribed by
Sample collection and analysis : Twenty-four-hour urine specimens were collected
at three speciÐc times. One specimen was collected before the start of the study,
while the patients were still receiving their usual diet (Diet 0). The other two urine
specimens were collected during the Ðnal 24 h of each study week (Diet 1 and Diet
The urinary excretion of HGA was analysed using gas chromatography of the
trimethylsilyl esters after ethyl acetate extraction (Duran et al 1987). The quantiÐed
HGA excretions were compared statistically using the paired t-test.
All patients with alkaptonuria had a normal neonatal period and development. On
sequential neurological examinations all patients were normal and showed no evidence
of neurological deÐcit. Growth before and after the initiation of dietary
protein restriction was normal with weight and length on the 45È55th centile of the
National Dutch Growth Statistics reference in all cases. The alkaptonuria patients
did not experience speciÐc problems at school.
The diagnosis of alkaptonuria was made at an average age of 1.4 years (2
monthsÈ4 years). The diagnosis was invariably suspected because of the discoloration
of the skin in the nappy region or the nappies themselves, or blackening of
urine on prolonged exposure to air. After diagnosis all patients were advised to
consume a diet with a protein restriction, usually 1.5 g/kg per day. Some patients
received administration of ascorbic acid. All patients had been consuming a proteinrestricted
diet (average 1.1 g/kg per day) since diagnosis. As diagnosis was conÐrmed
at dierent ages for each patient, varying from 2 months to 4 years of age, the
J. Inher. Metab. Dis. 21 (1998)
794 de Haas et al
period of protein restriction until start of the study varied for each patient. At the
beginning of the study, all patients younger than 12 years were still using a diet with
an average protein restriction of 1.2 g/kg per day. Although the Ðve patients older
than 12 years admitted that their compliance had not been very high in the recent
years before start of the study, analysis of their daily protein intake revealed an
average protein intake of 1.0 g/kg per day.
Figure 1 summarizes the important clinical and social parameters of alkaptonuria,
as recorded in speciÐc periods of age. Minor signs of abnormal pigmentation
were observed in all patients. Some children had a brown discoloration of the ear
helices, other had a blue pigmentation of the sclerae. Children younger than 6 years
showed a discoloration of the nappy region. The abnormal pigmentation tended to
aggravate with age, from 1 out of 9 for the 0È2 year-old children to a maximum of 4
out of 12 for the patients aged 6È18 years. Most abnormal pigmentation was
described as starting at an average age of 7È8 years.
As children grew up, increasing social problems were observed, especially behavioral
problems and difficulties with dietary compliance. The behavioral problems
mainly consisted of uncooperativeness without harmful consequences. The older
patients seemed to have more problems in acceptance of their disease and the therapeutic
consequences of trying to prevent ochronosis.
Social acceptance concerning dietary compliance emerged as the main problem.
The youngest children showed the best compliance. In general, these children were
Figure 1 Clinical signs of 16 alkaptonuric patients : cumulative number of patients for each
J. Inher. Metab. Dis. 21 (1998)
Dietary protein restriction in alkaptonuria 795
Table 1 Homogentisic acid excretion in 12 alkaptonuric patients
Diet 0 (usual diet) Diet 1 (low protein intake) Diet 2 (high protein intake)
Protein HGA Protein HGAa Protein HGAb
Age W eight (mmol/mmol (mmol/mmol (mmol/mmol
No. (years) (kg) (g) (g/kg) creatinine) (g) (g/kg) creatinine) (g) (g/kg) creatinine)
1 3 10.0 13.0 1.3 2.6 10.3 1.0 2.0 50.5 5.0 3.2
2 5 18.6 21.9 1.2 1.5 18.7 1.0 1.3 93.3 5.0 3.0
3 8 24.5 27.2 1.1 1.5 24.5 1.0 1.2 123.3 5.0 3.1
4 8 26.0 23.2 0.9 1.4 25.8 1.0 2.7 130.0 5.0 3.9
5 10 28.0 42.5 1.5 1.4 28.0 1.0 0.7 137.8 4.9 2.3
6 11 55.0 62.8 1.1 1.7 55.2 1.0 1.2 162.9 3.0 1.5
7 12 28.5 31.4 1.1 2.0 27.5 1.0 3.2 142.5 5.0 2.7
8 13 41.0 45.9 1.1 2.2 40.9 1.0 1.7 144.4 3.5 1.7
9 13 51.0 56.1 1.1 2.8 50.8 1.0 3.8 255.0 5.0 5.5
10 23 72.0 82.4 1.1 1.4 72.3 1.0 1.0 240.2 3.3 1.3
11 24 66.0 59.8 0.9 0.9 69.0 1.0 0.9 233.7 3.5 0.7
12 27 80.0 90.9 1.1 1.1 81.4 1.0 1.1 242.1 3.0 1.4
Mean value HGA excretion : a Diet 1, 1.73 mmol/mmol creatinine (SD 0.99) ; b Diet 2, 2.50 mmol/mmol creatinine (SD 1.34)
For patients under 12 years : HGA excretion with Diet 1 was signiÐcantly lower than the excretion with Diet 2: p\0.01
For patients older than 12 years : this dierence was not signiÐcant : p\0.193
J. Inher. Metab. Dis. 21 (1998)
796 de Haas et al
considered to be normal, having the usual behavioural struggles during adolescence,
augmented by having a restricted diet. The dietary compliance decreased to 1 out of
4 in subjects older than 18 years in comparison with the younger patients.
Signs of arthritis were not noticed in any of the patients (data not shown). As
complaints of arthritis in adults are known to be shown in spine, shoulders and
knees, the physical examination of patients focused mainly on these aspects. One
12-year-old patient complained of pain in the spine and knees, which was not
accompanied by radiographic evidence of ochronosis. These complaints disappeared
as the patient grew up.
When reviewing the excretion of homogentisic acid over the past few years versus
the presumed stable intake of protein, measured at outpatient clinic visits, we could
not Ðnd a stable level in the excretion of HGA. As an example, patient 10 excreted
0.7È2.3 mmol homogentisic acid/mmol creatinine over a 10-year period (mean 1.35,
SD 0.44). In healthy persons, HGA in the urine is undetectable. The HGA excretion
was signiÐcantly (p\0.01) lower (1.73 mmol/mmol creatinine, SD 0.99) with Diet 1
(1 g/kg per day protein) than the excretion (2.5 mmol/mmol creatinine, SD 1.34) with
Diet 2 (5 g/kg per day protein) in the patients under 12 years of age (Table 1). The
protein intake had minor or no eect on the urinary excretion of HGA in patients
older than 12 years.
A careful review of the medical histories of our 16 patients with alkaptonuria has
revealed no major clinical manifestations of complications of this disease during
childhood or adolescence. All patients showed normal growth and development.
The relationship between the level of pigmentation and duration of treatment with
protein restriction has not previously been studied. All our patients were consuming
a diet with protein restriction from the time of diagnosis. They all had minor signs
of abnormal pigmentation. We wonder whether this modest degree of abnormal
pigmentation can be considered a result of the protein restriction. Social problems,
i.e. those concerning acceptance of the fairly strict dietary regimen resulting in a
lower dietary compliance, became manifest in a number of patients over the age of
12 years. In relation to this, behavioural problems such as uncooperativeness
without harmful consequences were observed.
There has been much discussion concerning the best treatment for alkaptonuria
and prevention of its complications. The treatment focuses upon three aspects : correction
of the metabolic defect, restriction of the production of HGA, and prevention
of complications. At present, replacement of the defective enzyme is not feasible.
Several therapies have been suggested for the reduction of HGA excretion. One of
these proposals is treatment with vitamin C. Although ascorbic acid is known to
reduce the oxidation of HGA in vitro, it is very difficult to measure a positive eect
Protein restriction has been proposed as the most logical form of treatment. This
is based on the hypothesis that a diet with restriction of protein will lead to a lower
excretion of HGA, an intermediary metabolite in phenylalanine and tyrosine cata-
J. Inher. Metab. Dis. 21 (1998)
Dietary protein restriction in alkaptonuria 797
bolism. Assuming that the excretion of HGA is a parameter for the severity of
alkaptonuria, this would mean that restriction of protein may have a positive eect
on the clinical complications of alkaptonuria. In an attempt to prove this theory, we
investigated the 24-h urine HGA excretion after a protein intake of 1 g/kg per day
for 1 week versus the excretion after a protein intake of 5 g/kg per day for 1 week.
Minimal protein requirements for children, advised by the Dutch Ministry of
Health, are as follows : young children (1È4 years of age) require 1.25 g protein/kg
per day, while this amount of protein decreases to an average requirement of 0.70 g/
kg per day for children aged 16È19 years. The World Health Organization recommends
a dietary protein intake of at least 1.4 g/kg per day for all growing children.
Our results have shown that there is a signiÐcant decrease in the excretion of
HGA after a diet with a protein intake of 1 g/kg per day. This result conÐrms the
proposed hypothesis. The amount of HGA in urine related to dietary protein may
be inÑuenced by the age of the patient. The total group of patients can be divided
into two subgroups, namely patients under 12 years and patients over 12 years of
age. All patients received a low-protein diet (Diet 1) followed by an age-corrected
high-protein diet (5 and 3 g/kg per day, respectively). The subgroup of young children
clearly had an eect from increasing the protein intake, whereas the older ones
did not. From this we conclude that protein restriction in alkaptonuria in older
patients is probably useless.
We have documented that protein restriction in young alkaptonuric patients
resulted in a lower HGA production. In considering the hypothesis that the formation
of ochronotic pigment is equivalent to the production rate of HGA, we may
conclude that our patients, who were all treated from infancy or early childhood,
will attain a lower degree of deposit formation when they reach adulthood than
would be expected had they not been treated. Assuming that the signs of arthritis
are directly related to the amount of ochronotic pigment deposition in joints, it
seems valid to conclude that the low-protein diet will postpone the arthritic changes
by one or perhaps two decades. Continuation of the diet through adulthood is
advisable. However, as the patients are not ill at this particular moment and are not
able to judge the consequences of not taking this diet, it has appeared to be difficult
to convince the present group of adult patients of this need.
Bosacco SJ, Fye M, Finn CA (1991) Alkaptonuria and ochronosis. Orthopedics 14: 394È396.
Duran M, Ketting D, Bruinvis L, Dorland L, Wadman SK, Bakkeren JAJM (1987) Urinary
excretion of 2-methyl-2,3-butanediol and 2,3-pentanediol in patients with disorders of propionate
and methylmalonate metabolism. Clin Chim Acta 165: 197È204.
Gaines JJ (1989) The pathology of alkaptonuric ochronosis. Hum Pathol 20: 40È46.
Konttinen YT, Hoikka V, Landtman M, et al (1989) Ochronosis: a report of a case and a
review of literature. Clin Exp Rheumatol 7: 435È444.
La Du BN, Zonnoni VG, Laster L, Seegmiller JE (1958) The nature of the defect in tyrosine
metabolism in alkaptonuria. J Biol Chem 230: 251.
Lustberg TJ, Schulman SD, Seegmiller JE (1970) Decreased binding of 14C-homogentisic
acid induced by ascorbic acid in connective tissues of rats with experimental alkaptonuria.
Nature 228: 770È771.
J. Inher. Metab. Dis. 21 (1998)
798 de Haas et al
Martin JP, Batko B (1987) Homogentisic acid autoxidation and oxygen radical generation :
implications for the etiology of alkaptonuric patients. Free Radicals Biol Med 3: 241È250.
Montagutelli X, Lalouette A, Coude M, Kamoun P, Forest M, Guenet JL (1994) Aku, a
mutation of the mouse homologous to human alkaptonuria, maps to chromosome 16.
Genomics 19: 9È11.
OÏBrien WM, La Du BN, Bunim JJ (1963) Biochemical, pathologic and clinical aspects of
alkaptonuria, ochronosis and ochronotic arthropathy. Review of world literature (1584È
1962). Am J Med 34: 813È838.
Sealock RR, Galdston M, Steel JM (1940) Administration of ascorbic acid to an alkaptonuria
patient. Proc Soc Exp Biol Med 44: 580È583.
Wol JA, Barshop B, Nyhan WL, et al (1989) Eects of ascorbic acid in alkaptonuria: alterations
in benzoquinone acetic acid and an ontogenic eect in infancy. Pediatr Res 26:
SOCIETY FOR THE STUDY OF
INBORN ERRORS OF METABOLISM
The SSIEM was founded in 1963 by a small group in the North of England but now
has more than 70% of its members outside the UK. The aim of the Society is to
promote the exchange of ideas between professional workers in dierent disciplines
who are interested in inherited metabolic disorders. This aim is pursued in scientiÐc
meetings and publications.
The Society holds an annual symposium concentrating on dierent topics each
year with facilities for poster presentations. There is always a clinical aspect as well
as a laboratory component. The meeting is organized so that there is ample time for
informal discussion ; this feature has allowed the formation of a network of contacts
throughout the world. The international and multidisciplinary approach is also
reÑected in the Journal of Inherited Metabolic Disease.
If you are interested in joining the SSIEM then contact the Treasurer : Dr. J. R.
Bonham, Department of Paediatric Chemical Pathology, Sheffield ChildrenÏs Hospital
(NHS) Trust, Sheffield, S10 2TH, UK. The subscription includes the 8 issues
of the Journal of Inherited Metabolic Disease as well as the regular circulation of
The SSIEM web site is on http ://www.ssiem.org.uk