Hepatic peroxisomes are smaller in primary hyperoxaluria type I (PH I). (cytochemistry and morphometry)

Micron and Microscopica Ada, Vol. 20, No. 2. pp.125—126, 1989. Printed in Great Britain.

0739—6260/89$3.00 + 0.00 Pergamon Press plc

HEPATIC PEROXISOPES ARE SMALLER IN PRIMARY HYPEROXALURIA TYPE I (PH 1). MORPHOPETRY) *

De Craemer, D., Rickaert, F.

(CYTOCHEMISTRY AND

** ,

Wanders, R.J.A.

& Roels, F.

Human Anatomy, Vrije Universiteit Brussel, 1090 Brussels, Belgium * Anatomie Pathologique, H8pital Erasme, Université Libre de Bruxelles, Belgium ** Department Pediatrics, A.M.C. Amsterdam, the Netherlands.

Primary hyperoxaluria type I (PH I) is an autosomal recessive metabolic disorder characterized by increased synthesis of oxalate leading to nephrocalcinosi5 and systematic oxalosis. It is caused by the failure to metabolize properly glyoxylate by alanine:glyoxylate aminotransferase which in man is normally localized in the hepatic peroxisomes. In PH 1 this enzyme is deficient, and glyoxylate is oxidized in the cytoplasm to oxalate by lactate dehydrogenase. We examined two specimens of liver, one of a girl of 15 years with renal failure, and one of a boy who died at 8 months (Wanders et al., 1987). Specimen 1 was fixed in 2% glutaraldehyde; specimen 2 was kept unfixed at —80°C for several months, and frozen sections were immersed before thawing in buffered formaldehyde + 1% CaC1 2 containing DM50 as a cryoprotective. As controls, samples of 4 normal livers were fixed in 2% glutaraldehyde or it, 4% formaldehyde. All were stained for catalase activity with diaminobenzidine (Roels et al., 1988). In the liver of both patients, the reaction was weak but peroxisomes were visible by light microscopy. Under the electron microscope, the peroxisomes appeared as electron—dense organelles (Fig. la,b). Using a semi—automated IBAS (Kontron), morphometry was carried out on micrographs taken at random. Final magnification was approximately 20,000 and2calibrated with a grating of 2157 lines/mm. In each individual liver, more than 1,000 pm cytoplasm was investigated. The results are given in the table below. 3) V (%) number d—circle (pm) N (pm~ Peroxisomes meart+/—SEM max m~an+/—SEM meXn+/—SEM Patient 1

125

0.43+/—0.01

0.64

0.093

0.60

Patient 2

51

0.33+/—0.01

0.53

0.136

0.38

0.53÷/—0.01

0.85

0.065+/—0.010

0.87+/—0.11

0.53+/—0.02

0.90

0.081+/—0.003

1.05+1—0.14

3 controls 194 (fixed in G) 2 controls 257 (fixed in F)

D—circle is the diameter of the circle with the same area; volume density (V is expressed as the fraction of total cellular volume; numerical density (N ). as the numb~r of organelles per volume. V and Nv were calculated according to Weibel ~‘1969).Mean as well as maximal size of peroY
125

D. De Craemer et al.

126

Smaller peroxisomes were measured manually in 6 PH livers by Danpure and co—workers (lancu & Danpure, 1987; Danpure et al., 1989). However, no cytochemical identification with diaminobenzidine was performed nor was the volume density investigated. In the table below, our results are compared with theirs. d—max(pm) mean+/—SEM max

frequency mean

Patient 1

0.47+/—0.01

0.70

5.1

Patient 2

0.38+/—0.01

0.53

5.5

4 PH—patients (Iancu & Danpure) 2 PH—patients (Danpure et al.)

0.38

—

0.48 5.9

—

9.0

Dmax is the maximu~diameter of the organdies. Frequency is expressed as the number of peroxisomes/100 pm cytoplasm(i.e. excluding nuclei and sinusoidal spaces). Despite the differences in method, their results are similar to ours. Danpure et al. (1989) found no significant differences in peroxisome frequency between PH patients and controls. However, lancu & Danpure (1987) found a decrease in the ratio of the number of peroxisomes to the number of mitochondria. This may be due to changes in mitochondrial compartment. Both livers contained iron, recognized as electron—dense particles, and by the Pens stain. In specimen 1 it was present in large amounts in both parenchymal and Kupffer cells, with the highest concentration in the latter; in specimen 2 it was only present in parenchym. This indicates that in the older child, part of the iron originated in transfusions and hemodialysis, but that PH in itself causes Fe—accumulation in the parenchyma.

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Fig 1: Catalase staining of peroxisomes (arrowheads) in patient 1 (Fig a) and 2 (Fig b). In both livers, a close relationship between the endoplasmic reticulum and the peroxisomes is seen as usuai.Both livers also contain siderosomes (5). In patient 2, mitochondria (M) are damaged due to freezing before fixation. RER is dilated. Peroxisomal matrix also demonstrates loss of material. Bar: 1pm. References: Danpure et al., 3. Cell Biol. 1989, 108: 1345. — Geerts et al., Histochemistry 1984, 80: 339. — lancu & Danpure, 3. Inher. Metab. Dis. 1987, 10: 330. — Roels et al., Virch. Arch. A 1988, 413: 275. — Wanders et al., Clin. Chim. Acta 1987, 165: 331. — Weibel, Int. Rev. Cytol., 1969, 26: 236. Supported by special funds from the Belgian Ministry for Research Policy and Research Council V.U.B.