Copper deficiency secondary to a copper transport defect: A new copper metabolic disturbance

Copper Alan

Deficiency

L. Buchman,

Secondary to a Copper Transport Metabolic Disturbance

Carl L. Keen, Dennis

Harry V. Vinters,

Rodgerson,

Jorge

Edward

Vargas,

Harris,

Anthony

Defect:

Harry T. Chugani,

Verity,

and Marvin

A New Copper Bronwyn

Bateman,

Ament

We describe a 21-year-old man who developed copper deficiency manifested as a demyelinating neuropathy, chronic intestinal pseudo-obstruction, osteoporosis, testicular failure, retinal degeneration, and cardiomyopathy with a totiuous aorta. His serum copper was low and did not increase despite administration of large doses of intravenous copper sulfate. The ceruloplasmin level as measured by an antibody technique was normal, yet ceruloplasmin (Cp) oxidase activity was very low. The Cp amino acid sequence was normal. This suggests that the copper deficiency was caused by a defect in hepatic processing of copper for incorporation into Cp. Copyright 0 1994 by WA Saunders Company

T

HE PATIENT WAS a 21-year-old man of Asian and Fijian Indian descent who developed copper deficiency manifested as a demyelination neuropathy, chronic intestinal pseudo-obstruction, osteoporosis, testicular failure, retinal degeneration, and cardiomyopathy with a tortuous aorta. CASE REPORT Cerebellar

Ataxia Development

Progressive ataxia with limb incoordination developed at age 13 years when the patient noted difficulty with fine motor movements required for a typing class. The patient’s height was now normal for his age, although his weight remained below average. At age 16 years (March 1987). the patient’s muscle strength was diffusely and mildly diminished (4+ /5 on the standard neurological scale) and his eyelids were slightly ptotic. Finger-to-nose and heel-to-shin tests were moderately abnormal. Proprioception in both hands was mildly impaired. Deep tendon reflexes were normal. The patient’s mental status was normal. His gait had become progressively more ataxic over a severalmonth period, and he developed an acute transient hearing loss. Shortly thereafter, the patient developed a diffuse headache associated with burning pain on the right side of his body. He had a grand mal seizure, and phenytoin was administered. Serum electrolytes and albumin were normal. Later, the patient was maintained on valproic acid without seizure recurrence. Brain magnetic resonance imaging ([MRI] T1 and Tl weighted images) revealed a single region of increased signal density in the deep white matter of each cerebral hemisphere (periventricular and semi-ovale regions; Fig 1). Both serum phenytoin and valproic acid were maintained in the therapeutic range. Immunologic tests for toxoplasmosis, rubella, human immunodeficiency virus, IgG, IgA, CD4 and CDB. erythro-

From the Section of Gastroenterology, Baylor College of Medicine, Houston, TX; the Divisions of Pediatric Gastroenterology and Nutrition, Neuropathology Pediatric Neurology, Pediatric Ophthalmology and Clinical Chemisty, UCLA Medical Center, Los Angeles, CA; the Department of Nutrition, University of California at Davis, Davis, CA; and the Department of Biochemistry and Biophysics, Texas A&M Universiiy, College Station, TX. Submitted May 3, 1993; accepted March I, 1994. Address reprint requests to Alan L. Buchman, MD, MSPH, Baylor College of Medicine, The Methodist Hospital, Section of Gastroenterology, 6550 Fannin, Smith Tower 1122, Houston, TX 77030. Copyright 0 1994 by U?B. Saunders Company 00260495/9414312-0002$03.0010 1462

cyte sedimentation rate, antinuclear antibody, and rheumatoid factor were all normal. Lipoprotein electrophoresis, serum verylong-chain fatty acids, serum phytanic acid, myaline basic protein, and the plasma amino acid profile were all normal. Trypsin-Giemsa chromosome-banding analysis was normal. Urine tests for arylsulfatase. aminolevulnic acid, volatile and nonvolatile organic acids, acetic acid, aromatic acids, keto-acids, methylmelonic acid, cyanide nitroprusside, Benedict-Clinitest, dinitrophenyl hydrazine, heavy metals, homovanillic acid, vanillylmandelic acid, and toxicology were all normal. An electroencephalogram was abnormal: low-voltage waves were observed in the anterior portion of the brain, and a 7-8 cps rhythm with some intermixed 5-7 cps waves were observed in the posterior portion. Nerve-conduction study showed markedly reduced amplitudes of motor units produced in the lower extremities and borderline slowing of conduction velocity in the right median nerve. An electromyogram was normal. Cerebral spinal fluid studies were normal, with the exception of an elevated IgG synthesis rate (46.3 and 56.5 mg/dL; normal, 9.9 to 3.3 mg/dL). The IgG level was normal. In July 1987. a brain biopsy of the right frontal lobe was performed because of consideration of subacute sclerosing panencephalitis and multiple sclerosis. Examination by light and electron microscopy revealed no significant abnormality apart from scattered astrocytes. In August 1987, the patient became encephalopathic presumably secondary to hepatic toxicity associated with valproic acid, which was confirmed by percutaneous liver biopsy. Hepatic copper and urine copper were normal (Table 1). Valproic acid was discontinued, and the toxicity resolved: no further anticonvulsant medication was administered. The patient’s mental status became normal. Neurological examination revealed a fine motor tremor. The patient began to experience occasional urinary incontinence. By May 1989, the patient could stand but was no longer able to walk. He had difficulty sitting upright in a chair, and by June 1990 he lost head control intermittently. A nerve-conduction study at this time showed no activity in the right or left peroneal. tibial, or sural nerve. A sural nerve biopsy (Fig 2) showed profound loss of myelinated fibers with a virtual absence of any large myelinated fibers. Schwann cell hyperplasia was observed. A muscle biopsy showed many necrotic fibers, scattered acid phosphatase-positive fibers (indicating the presence of histiocytes), and rare alkaline phosphatase fibers. Positive adenosine triphosphatase staining showed a moderate degree of fiber-type grouping. Nicotinamide adenine dinucleotide and SDH staining showed prominent central clearing in many type 1 fibers. An oil red stain showed increased lipid in several fibers, especially those that were hypertrophic. Gomori trichrome staining showed rare ragged red-like fibers. Electron microscopy showed many fibers containing markedly disorganized and abnormally arranged myofilaments. Metabolism, Vol43, No 12 (December), 1994: pp 1462-1469

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COPPER DEFICIENCY CAUSED BY TRANSPORT DEFECT

Fleischer rings; the drusen-like deposits persisted in each macula. An arcus juvenilis was noted in each cornea. The etiology for this was uncertain; lipoprotein electrophoresis was normal. An electroretinogram was abnormal, with cone function affected more than rod function. A conjunctival biopsy showed nonspecific changes. Some fibroblastic mitochondria appeared slightly tortuous and dilated.

Development of Pseudo-Obstruction The patient had experienced occasional cyclic vomiting associated with abdominal pain and distention since age 10 that interfered with his school performance, although even as an infant he would vomit his formula and later spilled food from his utensils while eating. At this time, the patient’s growth was in the 5th to 10th percentile and his weight was below the 5th percentile. Because of persistent vomiting, a gastrointestinal series was obtained that showed a dilated duodenal bulb with a normal small bowel, although the transit time was slightly delayed at 4.5 hours. A 99Tc-labeled solid gastric-emptying study showed 76% activity remaining in the stomach at 90 minutes (normal, 1.50% at 90 minutes). Pseudo-obstruction was diagnosed on a clinical basis, and this was also thought to be the origin of the patient’s occasional urinary incontinence. During the following 2 years, intermittent vomiting and abdominal distention continued. The patient became gradually malnourished, and a percutaneous endoscopic gastrostomy tube was placed for feeding. Because of frequent exacerbations of intestinal pseudoobstruction and poor gastric emptying, he was started on total parenteral nutrition (TPN). This included zinc 4 mgid. copper 2 mgid, selenium 20 kg/d, chromium 10 hgid. and multivitamins. A serum Cu level measured using inductively coupled plasma emission spectroscopy’.? was low before beginning TPN (Table I): serum Zn was 59 FgidL (normal, 60 to 130). and plasma Se was 41 ng/mL (normal, 95 to 165). The patient was administered a trial of domperidone for the pseudo-obstruction without symptom relief. In September 1989, a microcytic hypochromic anemia due to iron deficiency was discovered based on a hemoglobin of 8.3 g/dL. hematocrit of 27.3%. mean corpuscular volume of 74 fL. reticulocyte count of less than 140, serum iron of 15 mEq/dL, and total iron-binding capacity of 492 FgimL. Previous hemoglobin and hematocrit 4 years earlier were 12.4 g/dL and 37.X, respectively. The white blood cell count and differential were normal. Serum vitamin B,l and folate were normal. Iron dextran was administered

Fig 1. T, weighted brain MRI showing increased signal density in the deep white matter of each hemisphere (arrows).

Abundant subsarcolemmal glycogen was seen. Mitochondria showed no abnormal patterns or cristae. despite the presence of ragged red fibers on histochemical study.

Retinal Degeneration The patient’s visual acuity became markedly abnormal between ages 14 and 16 and continued progressive impairment until the patient became nearly blind at age 19. A color vision defect was detected using pseudoisochromic plates. Visual and brain stem evoked responses were abnormal; conduction times of the visual evoked responses were delayed. Fundoscopic examination revealed mascular degeneration with drusen-like deposits. On slit-lamp biomicroscopy, there was no evidence of KayserTable 1. Cu Concentrations

-

-4ge

Levels in Various Body Compartments Urine, pg124 h

Liver, pg/g

(norm, 70-155)

(norm, 15.50)

(norm, 10-35)

15

17 (August 1987)

4ge 19 (May 1989)

41

4ge 20 (June 1990) 2 wk later

51 49

July 1990 1 wk later

51 73.0

2 wk later

98.0

-

Gastrostomy, w/24

h

Cp, mg/dL

IV cu

(norm. 18.39)

intake, mgid

16

21

2 6 12 3,824

25

24

21.5

24 2

2,458

0

4,330

after penicillamine

NA

24

144

667

4ge 21 (September 1990) Before penicillamine 24 h

and Cu Intake in the Patient at Different Ages

Serum, bg/dL

0

2 wk after trientene hydrochloride started 3 d after trientene hydrochloride discon-

166

5,596

tinued 2 wk after trientene hydrochloride discon-

138

6,324

0

4,330

0

tinued Abbreviation: NA, not available.

168

39

0

1464

BUCHMAN ET AL

Fig 2. (A) Plastic-embedded sections of the peripheral nerve biopsy (stained with toluidine blue) show profound loss of myelinated fibers. (Original magnification x570.) (6) Normal peripheral nerve biopsy (toluidine blue). (Original magnification x80.)

intravenously with a resultant increase‘in the reticulocyte count to 4.8% and a gradual reduction of the anemia. Also at this time, the patient had a normal metapyrone test. He was then treated with prednisone 40 mg daily for 2 weeks because of continued neurological deterioration, but did not improve.

Testicular Failure As part of a battery of endocrine tests, the patient was found to have low testosterone levels (total testosterone, 6.0 ng/dL [normal, 350 to 1,030]; free testosterone, 0.4 pg/mL [normal, 52 to 2801). Sex hormone-binding globulin was normal. All other endocrine tests, including thyroid and prolactin, were normal.

Cardiomyopathy In January 1990, an echocardiogram showed normal left ventricular size but inferoapical hypokinesis to akinesis, with an estimated left ventricular ejection fraction of 50% to 55%. The descending thoracic aorta was ectatic throughout its course. An electrocardiogram was normal.

Osteoporosis In March 1990, computerized tomography bone density scan of the lumbar-sacral spine showed an average of 52 mg/mL (normal, 190.7 ? 28.5 for age). A subsequent bone biopsy performed on the

1465

COPPER DEFICIENCY CAUSED BY TRANSPORT DEFECT

anterior iliac crest following double-tetracycline labeling showed substantial reduction in the volume of cancellous bone, as well as in cortical bone thickness. A moderate increase in osteoids with relatively few osteoblasts, resorption lacunae. and only occasional osteoclasts was seen. Histochemical staining for aluminum was negative, and only occasional double-tetracycline labels were identified. The separation between double-tetracycline labels and label length were both reduced.

C’LCDeficiency

In July 1990. the patient’s serum Cu (Table 1) was still low despite Cu-containing TPN, although serum ceruloplasmin (Cp) was normal (Table 1): his TPN was supplemented with additional Cu. The patient had normal hair texture and no hypopigmentation. When the serum Cu failed to increase, additional Cu was added to the TPN in a stepwise fashion. Plasma Cp oxidase activity as determined by the Rice procedure’ was 19.6 IU/L (normal controls. 40 to 95). Polymerase chain reaction of the coding sequence for the patient’s Cp was performed by isolating RNA (15 up) from a liver biopsy specimen and performing first-strand synthesis using oligo (dT).“,’ The polymerase chain reaction fragments obtained permitted sequencing of only 45% of the Cp RNA; however, no abnormality was seen. The patient’s mother had a Cp activity of 44.8 IUIL, serum Cu concentration of 184 ug/dL. and serum Cp concentration of 50 mg/dL. His father had a Cp activity of ‘2.30 IUIL, serum Cu concentration of 144 ug/dL, and serum Cp concentration of 34 mg/dL. The patient’s only sibling, a sister. declined phlebotomy. Serum Cp concentration was determined by nephrotomy.” At the time the serum Cu normalized, lactate dehydrogenase was 287 U/L (normal, 100 to 180) and haptoglobin was 318 mg/dL (normal, 35 to 224), although no evidence of hernolysis was seen on the peripheral smear. A red blood cell superoxide dismutase (SOD) concentration (measured according to :he procedure described by Marklund’) at this time was 0.80 ug, mg hemoglobin (normal, 0.86 2 0.02). The patient developed jaundice rapidly, and a liver biopsy was performed. It showed mild pot tal lymphocytic infiltration with fibrous expansion extending into the lobules. Severe fatty change and hepatocellular swelling wa< seen. Minimal cholestasis was present. The hepatic Cu concentration. measured by plasma emission spectroscopy followmg tissue digestion in concentrated nitric acid, was elevated significantly when compared with National Bureau of Standards t NBS) bovine liver standards and with the initial biopsy measurement (Table 1). Serum vitamin A, Bh, and E. as well as serum zinc concentration. were normal. Plasma Se remained low (74 ng/mL). but was normalized by using a maintence dose of 110 pg daily in the TPN. Positron-emission tomography (PET) scanning of cerebral glucose metabolism revealed decreased uptake of the tracer 2-deoxy2[“F]Ruoro-u-glucose in the occipital cortex. but normal activity in tht basal ganglia (Fy-5; Fig 3). Because of the excessive hepatic Cu deposition noted on the second liver- biopsy, all Cu was removed from the patient’s TPN, and penicillamine was started at a dose of 750 mg daily. Two weeks later. fever occurred along with severe hemolytic Coombs-positive m:mia. A chest computed tomography scan revealed mediastinal lipamatosis. No etiology for the fever aside from penicillamine could be identified. and it was therefore discontinued. Hepatic transaminares remained essentially unchanged during this period, although the total bilirubin increased as high as 13.4 mg/dL. Prothrombin time remained normal. Because of his poor tolerance of penicillamine. trientene hydrochloride was started at a dose of 1,000 mg daily. Tubular reabsorption of phosphate was 56% (mnmal. > 78%).

4

)_

Fii 3. PET wan of central glucose utilization showing decreased glucose metabolism in the occipitotemporal regions bilaterally (large arrows). The normal uptake pattern in the lenticular nuclei (small arrows) is in contrast to the decreased glucose utilization typically seen in Wilson’s disease.*

As with the penicillamine, fever occurred with the trientene hydrochloride and it was discontinued. Serum Cu remained increased, but were within the range of normal values. However, low-grade hemolysis continued. Hepatic transaminases showed a moderate degree of spontaneous improvement, and the hemolysis resolved. The fever persisted despite broad-spectrum antibiotic coverage and subsequent discontinuation of all medication. The patient experienced a cardiac arrest thought to be secondary to septic shock based on a positive urine culture, although blood cultures remained negative. No previous cardiac arrhythmias had been identified. One week later he died; an autopsy was refused. DISCUSSION

We describe a young patient who developed manifestations of Cu deficiency before institution of TPN. which persisted despite usual Cu supplementation with TPN. We demonstrated Cu deficiency despite a lack of the previously described hallmarks of the disease-namely neutropenia and anemia. Although anemia was present, it was largely but not necessarily completely secondary to iron deficiency, which was treated. Given the observation that Cp oxidase activity was low despite normal serum Cp and apparently normal Cp structure, we believe that the primary defect involved an error in the transport of Cu into apolipoprotein Cp. Thus, normalization of serum Cu with supplemental intravenous Cu would not be achieved. Since the Cu did not bind to Cp, we suspect that it circulated predominantly in the plasma either largely bound to albumin or amino acids or in the free form. Biliary Cu excretion was not measured because the patient had only two bowel movements weekly and none during the 24-hour urine and gastrostomy tube collections. Although our patient had an impressive accumulation of hepatic Cu on the second liver biopsy, the findings of normal hepatic Cu at the first liver biopsy, the absence of Kayser-Fleischer rings, the presence of a peripheral demyelinating neuropathy, and the PET scan results preclude the diagnosis of Wilson’s disease in our patient. He had normal glucose metabolism in the basal ganglia on the PET scan, which is distinct from that seen in Wilson’s disease where hypermetabolism in the putamen is a prominent feature.8 Neifakh et al4 have described reductions in Cp oxidase activity of 30% and 35%. respectively, in two

1466

patients with Wilson’s disease; our patient had a 51% decrease. Accumulation of Cu in the renal tubules (similar to that seen in Wilson’s diseasegJ0) is the most likely cause of the renal tubular abnormality in our patient as manifested by the decreased tubular reabsorption of phosphate. There was no aminoaciduria or glucosuria suggestive of Fanconi’s syndrome.9 The recurrence of Se deficiency once the patient had received the extra Cu supplementation may have reflected Cu “overflow” from the liver with deposition in the renal tubules, and a subsequent increase in the tubular damage with a resultant increase in urinary Se secretion. The occurrence of massive hemolysis following penicillamine therapy was presumably the result of the release of non-Cp-bound Cu as indicated by the high-normal Cu concentrations. These Cu levels persisted long after penicillamine discontinuation, consistent with an overflow of Cu from the hepatocytes.” In normal subjects Cu is rapidly incorporated into Cp, thereby reducing the amount available for renal excretion.12 The gradual decrease over time in Cp protein as measured by antibody was probably a reflection of decreasing hepatic function following Cu loading; when Cu was released from the liver, Cp production increased. Our patient differed from those suffering with Menkes’ syndrome in that the hepatic Cu content was not low on the initial biopsy,13 his hair was normal, and no hypopigmentation or hypothermia was evident.9J4 Although the initial serum Cu concentration was obtained only shortly before initiation of TPN, it is likely that the Cu deficiency began early in life and was the basis of his neurologic degeneration. However, the patient never demonstrated the neutropenia commonly described in Cu deficiency,‘j and his anemia was corrected with iron supplementation alone. Nevertheless, Cu deficiency is a likely explanation for several of this patient’s major deficits including the demyelinating neuropathy, osteoporosis, testicular failure, tortuous aorta, cardiomyopathy, and retinal degeneration. We cannot explain the elevated cerebrospinal fluid IgG synthesis. It was surprising that despite the cerebral white matter abnormalities seen on several brain MRI scans suggestive of demyelination, normal myelinated fibers were found in brain biopsy. Involvement of the white matter is described in Menkes’ syndrome,16,*7 and demyelination or myelination abnormalities have been described in Cu-deficient rats.1s-20 Neuronal loss secondary to extensive demyelination in the cerebellum and spinal tracts in lambs with swayback or enzootic ataxia has been described.2’-22 Ataxia with cerebellar neuronal loss has also been found in the brindled mouse mutant, a neurological mutant mouse with a deficiency in Cu transport,23 and in humans (Menkes’ disease).24 Deficiencies of cytochrome oxidase and of dopamine p-hydroxylase may account for the demyelination due to Cu deficiency.22~25~26 Our patient had only a frontal lobe biopsy, and it is possible that the myelin involvement was not as pronounced at the time of the brain biopsy and therefore may not have been apparent on the biopsy.

BUCHMAN ET AL

Although the MRI was used as a guide, it is possible that sampling error may have led to biopsy of relatively normal tissue. However, a later nerve biopsy showed clear evidence of massive demyelination. The nerve-conduction studies clearly document the progression of peripheral nerve demyelination. The patient’s intestinal pseudo-obstruction may have been an early manifestation of nerve demyelination. Osteoporosis was severe in our patient. It has been described in Cu-deficient sheep27,2* and in Cu-deficient children.29,30 Although long-term TPN is also associated with metabolic bone disease,31 our patient had received TPN for only 16 months before the bone biopsy. In addition, the bone disease associated with TPN is usually patchy osteomalacia. We believe that our patient’s osteoporosis was probably a consequence of inadequate collagen cross-linking caused by lysyl oxidase deficiency induced by Cu deficiency.32,33 Although our patient had testicular failure, he received replacement doses of testosterone that should have corrected any osteoporosis caused by androgen deficiency.34 Furthermore, there was no evidence on the bone biopsy of osteomalacia of vitamin D-deficiency origin. There was no evidence of hyperparathyroidism, since the parathyroid hormone level was normal. Considering that osteoporosis is one of several disorders in our patient that are associated with Cu deficiency, it is likely that it was at least one factor in the development of osteoporosis. Cu deficiency has been associated with low concentrations of plasma testosterone in sheep and rats.35.36 In addition, degeneration of spermatogenic cells and seminiferous tubule necrosis have been observed in rats fed a Cu-deficient diet.35,3h When Cu-deficient sheep were fed Cu-supplemented diets, hepatic Cu and plasma testosterone normalized.” In addition, Cu has been found to stimulate follicle-stimulating hormone release in cultured rat pituitary cells, as well as in vivo in rabbits.37.38 These data suggest that Cu deficiency may be associated with deficiency in luteinizing hormone and follicle-stimulating hormone release. It is unclear whether our patient had primary or secondary testicular failure. The elevated folliclestimulating hormone and prolactin following testosterone replacement may have been secondary to the domperidone. The elevated prolactin from May 1989 cannot be explained when the level was normal in April 1989 and no pituitary adenoma was seen on the MRI. Nishi et al40 have suggested a relationship between prolactin and Cu metabolism on the basis of decreased urinary Cu output in patients with prolactinomas as compared with controls. They found urinary Cu but not Zn increased to a level similar to control levels following removal of the prolactinoma. However, no significant change in serum Cu was detected in this study. Hansen et a141 described a patient with depression and truncal ataxia with low serum Cu and Cp and elevated serum prolactin, the later of which subsequently became normal. Tortuous large arteries including the aorta have been described in Menkes’ syndrome; arteries may show areas of intimal proliferation, and stenosis with frayed and fragmented elastic fibers with reduplication of the internal

COPPER DEFICIENCY CAUSED BY TRANSPORT

1467

DEFECT

elastic lamina. 14,1hAlthough autopsy was not performed on our patient, a tortuous aorta compressing the left atrium was seen on an ultrasound examination. Defects in myocardial contractility have been demonstrated in isolated perfused heart from Cu-deficient rats4* and may be caused by an abnormal collagen framework.43 Although our patient had a low serum Se at the time the echocardiogram was performed, his slightly low left ventricular ejection fraction may have reflected both Cu and Se deficiency. Although electrocardiogram abnormalities have been demonstrated in Cu-deficient rats.44 our patient did not manifest these changes. In addition, Cu-deficient rats generally exhibit cardiac hypertrophy. probably as a result of the anemia.42 Our patient may not have developed cardiac hypertrophy because he was anemic for a relatively short period of time. Ocular abnormalities have been associated with Cu deficiency.2”.~5~4” A reduction in the number of retinal ganglion cells with optic atrophy and demyelination have both been described in a pathologic study of Menkes’ syndromc.2”,j5 Abnormal visual evoked responses manifested by diminished amplitude of the major positive peak, as seen in the case described herein, have been reported in Mcnkes‘ syndrome as well .4h-48Abnormality of the electroretmogram has been described in a child with Menkes’ syndrome: the changes were not reversible with Cu supplemcntation.Jq We cannot explain the finding of arcus juvenilis. It may be a hallmark of the disorder of Cu metabolism we have described herein. The superoxide dismutase activity levels in our patient were only slightly lower than in cent-rols. However, it has been demonstrated that levels of erythrocyte superoxide dismutase can be maintained even when there is no measurable serum Cp. Y)Therefore, erythrocyte superoxide dismutase activity levels may be a relatively poor indicator of Cu deficiency. The disorder of Cu metabolism reported herein is to our knowledge unique. It is distinct from Wilson’s disease and Mcnkes’ syndrome, as well as from the unclassified cases described by Ono and Kurisaki,” Willvonsede et al,5? Goodwin-Austen et aL5’ and Haas et al.54 Our patient also IS distinct from patients with Kearns-Sayre syndromes5 in th:tt he did not manifest dementia, lactic acidosis, short stature, or characteristic electrocardiogram findings. There was no family history of diseases similar to myoclonic epilepsy with ragged red fibers or mitochondrial encephalopathy, encephalopathy, lactic acidosis, and stroke-like episodes.?’ Our patient had retinal degeneration, which is not seen in myoclonic epilepsy with ragged red fibers or mltochondrial encephalopathy, encephalopathy, lactic acidosis. and stroke-like episodes.5s Recently, Simon et al56 described a syndrome they called POLIP (polyneuropathy, ophthalmoplegia, leukoencephalopathy, and intestinal

pseudo-obstruction). Although the laboratory data were incomplete in these five patients, the brain MRI findings in two patients and peripheral nerve demyelination in two patients are very similar to the case reported herein. Unfortunately, detailed information is not available for each patient and the reported laboratory data are incomplete. Two patients had deafness and the others had normal visual function and visual evoked potentials, which differ from the findings for our case, although these findings are nonspecific. One possibility is that various degrees of disease activity may have been present. Simon et al rejected the hypothesis that nutritional factors caused abnormalities in brain white matter, because the MRI abnormalities they described were present before the development of overt clinical malnutrition progressed despite parenteral nutritional supplementation. Unfortunately. of the myriad of laboratory tests performed on the various patients. Cu levels were undetermined. We suspect that at least two of the patients reported by Simon et al may have manifested the Cu metabolic disturbance we describe herein. Li et a15’ have also recently described a patient with pseudoobstruction and retinal abnormalities who bears some resemblance to our case. The cytochrome c oxidase concentration was low in that case and could have reflected Cu deficiency. Finally, Mehes and Petroviczsx described a child with a seizure disorder, hypochromic anemia caused by iron deficiency, low serum Cu, normal Cp concentration, and low urinary Cu excretion. Although no further investigation on this child was performed, these findings are similar to the case reported herein. Bannister and Wood5’ showed that a loss of human Cp oxidase activity was associated with the destruction of hi&dine residues in a one-to-one correspondence in vitro. These investigators suggested that the site responsible for the oxidase activity of Cp may contain an essential histidine residue. We were unable to detect such a deficit in the case reported herein, although only 45% of the patient’s Cp mRNA was sequenced. This suggested that the Cu deficiency was secondary to a defect in Cp-Cu transport rather than a chromosomal abnormality. In conclusion, we have described a patient who was Cu-deficient on the basis of defective Cp transport. He was unable to utilize intravenous Cu. The treatment for this newly described disorder remains unknown. The role abnormal Cu metabolism played in the patient’s final demise remains unclear. ACKNOWLEDGMENT

The authors thank Jonathan D. Gitlin. MD, for the Cp molecule sequencing, and Erman Sternlieb, MD, Angel Cardona, MD, Michel Philippart. MD, Roy Philby, PhD, and Gretchen Hill, PhD, for their helpful discussions.

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emis-

3. Rice EW: Standardization of ceruloplasmin activity in terms of international enzyme units. Oxidative formation of “Bandrous-

BUCHMAN

ki’s base” from p-phenylenediamine by ceruloplasmin. Anal Biothem 3:4X!-456, 1962 4. Neifakh SA, Vasiletz IM, Shavlosky MM: Molecular pathology of caeruloplasmin. Biochem Genet 6:231-238. 1972 5. Sadhu C, Gedamu I: Regulation of human metallothionein (MT) genes. J Biol Chem 263:2679-2684,1988 6. Pesce MA, Bodourian SH: Nephelometric measurement of ceruloplasmin with a centrifugal analyzer. Clin Chem 28:516-519, 1982 7. Marklund SL, Marklund G: Involvement of the superoxide anion radical in the auto oxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469-474.1974 8. Hawkins RA, Mazziotta JC, Phelps ME: Wilson’s disease studied with FDG and position emission tomography. Neurology 37:1707-1711,1987 9. Danks DM: Disorders of copper transport, in Stanbury JR, Wyngaarden JB, Fredrickson DS (eds): The Metabolic Basis of Inherited Disease (ed. 6). New York, NY, McGraw-Hill, 1989, pp 1411-1431 10. Wolff SM: Renal lesions in Wilson’s disease. Lancet 1:843845.1968 11. Roche-Sicot J, Benhamou JP: Acute intravascular hemolysis and acute liver failure associated with a first manifestation of Wilson’s disease. Ann Intern Med 86:301-303, 1977 12. Beam AG, Kunkel HG: Metabolic studies in Wilson’s disease using Cu@. J Lab Clin Med 45:623-631.1955 13. Danks DM, Stevens BJ, Campbell PE, et al: Menkes’ kinky hair syndrome. Lancet l:llOO-1102, 1972 14. Danks DM, Campbell PE. Stevens BJ, et al: Menkes’ kinky hair syndrome: An inherited defect in copper absorption with widespread effects. Pediatrics 50:188-201. 1972 15. Danks DM: Copper deficiency in humans. Annu Rev Nutr 8:235-257,1988 16. Aguilar MJ, Chadwick DL, Okuyama K, et al: Kinky hair disease. I. Clinical and pathological features. J Neuropathol Exp Neurol25:507-522, 1966 17. Danks DM, Cartwright E, Stevens BJ, et al: Menkes’ kinky hair disease: Further definition of the defect in copper transport. Science 170:1140-1142,1973 18. DiPaolo RV. Newberne PM: Copper deficiency and myelination in the newborn rat. Fed Proc 31:699,1972 (abstr) 19. DiPaolo RV. Kanfer JN, Newberne PM: Copper deficiency and the central nervous system. Myelination in the rat: Morphological and biochemical studies. J Neuropathol Exp Neurol33:221-236, 1974 20. Yajima K, Suzuki K: Neuronal degeneration in the brain of the brindled mouse a light microscope study. J Neuropathol Exp Neurol38:35-46, 1979 21. Barlow RM: Further observations in swayback. I. Transitional pathology. J Comp Pathol Ther 73:51-60, 1963 22. Smith RM. Osborne-White WS, O’dell BL: Cytochromes in brain mitochondria from lambs with enzootic ataxia. J Neurochem 26:1145-1148, 1976 23. Hunt DM: Primary defect in copper transport underlies mottled mutants in the mouse. Nature 249:852-854,1974 24. Garnica AD, Frias JL, Rennert OM: Menkes’ kinky hair syndrome: Is it a treatable disorder? Clin Genet 11:154-161. 1977 25. Prohaska JR: Functions of trace elements in brain metabolism. Physiol Rev 67:858-901, 1987 26. Prohaska JR, Smith TL: Effect of dietary or genetic copper deficiency on brain catecholamines, trace metals and enzymes in mice and rats. J Nutr 112:1706-1717, 1982 27. Suttle NF, Angus RW, Nisbet DI, et al: Osteoporosis in copper depleted lambs. J Comp Pathol82:93-97. 1972

ET AL

28. Whitelaw A, Armstrong RH, Evans CC, et al: A study of the effects of copper deficiency in Scottish blackface lambs on improved hill pasture. Vet Ret 104:455-460, 1979 29. Ashkenazi A, Levin S, Djaldetti M, et al: The syndrome of neonatal copper deficiency. Pediatrics 5:525-533, 1973 30. Yven P, Lin HJ, Hutchinson JH: Copper deficiency in a low birth weight infant. Arch Dis Child 54:553-555, 1979 31. Klein GL, Ament ME. Bluestone R, et al: Bone disease associated with total parenteral nutrition. Lancet 2:1041-1044, 1980 32. Royce PM, Camakaris J, Danks DM: Reduced lysyl oxidase activity in skin fibroblasts from patients with Menkes’ syndrome. Biochem J 192:579-586, 1980 33. Royce PM, Camakaris J, Mann JR, et al: Copper metabolism in mottled mouse mutants. The effect of copper therapy on lysyl oxidase activity in brindled (Mob’) mice. Biochem J 238:177183,1986 34. Finkelstein JS, Klibanski A, Neer RM, et al: Increases in bone density during treatment of men with idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 69:776-783, 1989 35. Van Niekerk FE, Van Niekerk CH: The influence of experimentally induced copper deficiency on the fertility of rams. I. Semen parameters and peripheral plasma androgen concentration. S Afr Vet Ver 60:28-31, 1989 36. Mehta V, Mehta S, Georgie GC, et al: Effect of dietary zinc and copper on peripheral blood cholesterol, testosterone and histomorphology of testes in rats, Indian J Exp Biol 27:469-471, 1989 37. Stemmer KL, Liu J: Interaction of aluminum with zinc and copper and its effects on pituitary-testicular axis: A histological study. Biomed Environ Sci 3:1-10. 1990 38. Pau FKY, Spies HG: Effects of cupric acetate on hypothalamic gonadotropin-releasing hormone release in intact and ovariectomized rabbits. Neuroendocrinology 43:197-204, 1986 39. Hazum E, Schwartz I: Copper induces luteinizing hormone release and desensitization of pituitary gonadotropes. Biochem Biophys Res Commun 136:417-425,1986 40. Nishi Y, Aihara K, Hatano S, et al: Concentrations of zinc, copper, manganese and selenium in blood and urine of patients with prolactinoma. Acta Paediatr Jpn 30:199-203, 1988 41. Hansen CR, Malecha M, Mackenzie TB, et al: Copper and zinc deficiencies in association with depression and neurological findings. Biol Psycho1 18:395-401, 1983 42. Prohaska JR. Heller LJ: Mechanical properties of the copper-deficient rat heart. J Nutr 112:2140-2150, 1982 43. Borg TK, Klevay LM, Gray RE, et al: Alteration in the connective tissue network of striated muscle in copper deficient rats. Mol Cell Cardiol 17:1173-1183, 1985 44. Viestenz KE, Klevay LM: A randomized trial of copper therapy in rats with electrocardiographic abnormalities due to copper deficiency. Am J Clin Nutr 35:258-266, 1982 45. Levy NS, Dawson WW. Rhodes BJ, et al: Ocular abnormalities in Menkes’ kinky hair syndrome. Am J Ophthalmol77:319-325, 1974 46. Billings DM, Degnan M: Kinky hair syndrome. Am J Dis Child 121:447-449, 1971 47. Singh S, Bresnan MJ: Menkes’ kinky hair syndrome (trichopoliodystrophy). Low copper levels in the blood, hair and urine. Am J Dis Child 125:572-578. 1973 48. French JH, Sherard ES: Trichopoliodystrophy. I. Report of case and hio-chemical studies. Arch Neurol26:229-244, 1972 49. Seelenfreund MH, Gartner S. Vinger PF: The ocular pathology of Menkes’ disease. Arch Ophthalmol80:718-720,1968 50. Alexander NM, Benson GD: Superoxide dismutase activity (erythrocuprein) in Wilson’s disease. Life Sci 16:1025-1032,1975

Increased

Susceptibility to Lipid Oxidation of Low-Density Lipoproteins Erythrocyte Membranes From Diabetic Patients

and

Rosa A. Rabini, Paolo Fumelli, R. Galassi, Nicole Dousset, Marina Taus, Gianna Ferretti, Laura Mazzanti, Giovanna Curatola, Marie L. Solera, and Pierre Valdiguik The aim of the present study was to determine if low-density lipoproteins (LDLs) and red blood cell (RBC) membranes from diabetic patients present an increased susceptibility to lipoperoxidation, which might be related to the increased incidence of atherosclerosis in diabetes. LDLs and RBC membranes were isolated from 11 insulin-dependent (IDDM) and 18 non-insulindependent diabetic (NIDDM) patients and exposed to a peroxidative stress by incubation with phenylhydrazine. The susceptibility to peroxidation was determined by measuring the production of thiobarbituric acid-reactive substances (TBARS) after the incubation. The following parameters were also evaluated: plasma glucose, triglycerides (TG), phospholipids (PL), total and high-density lipoprotein (HDL) cholesterol, apolipoprotein (ape) A-l, apo B, hemoglobin AI, (HbA,,), LDL PL and cholesterol, LDL fatty acid composition, and RBC membrane PL and cholesterol. Although they were apparently normolipidemit, diabetic patients showed an increased susceptibility to peroxidation in LDLs and erythrocyte membranes as compared with control subjects. The amount of arachidonic acid in LDLs and the PL concentration of RBC membranes from diabetic patients were significantly higher than in normal subjects. The increased lipoperoxidability of both RBC membranes and LDLs might play a central role in the pathogenesis of the vascular complications of diabetes mellitus. Copyright 10 1994 by W.B. Saunders Company

T

HE CENTRAL ROLE of lipid peroxidation in the pathogen&s of atherosclerosis has been suggested by several in vivo and in vitro studies.‘” The oxidized lowdensity lipoproteins (oxLDLs) are recognized by the scavengcr receptor of macrophages. with subsequent storage of cholesterol in their cytoplasm in the form of lipid droplets and transformation of macrophages into foam cells.4 Moreover, oxLDLs are cytotoxic to endothelial cells and might therefore directly determine endothelial damage.’ Oxidative modification of LDL seems to occur primarily in the intima of the arterial wall,h as suggested by the observation that endothelial cells “in vitro” are able to oxidize LDL.’ ox-LDL can be locally taken up by the macrophages or rapidly cleared from the circulation by sinusoidal cells in the liver, spleen, and bone marr0w.s On the basis of these observations, it can be supposed that the determination of plasma levels of circulating ox-LDL might not give reliable information on the events developing at the level of the arterial wall. Therefore, the assay of LDL susceptibility to peroxidation using in vitro oxidative stress has been proposed as a tool to verify the predisposition to the “in vivo” development of ox-LDL inside the arterial N all.4~l” The aim of the present study was to determine if patients affected

by insulin-dependent

dependent

(NIDDM)

creased

susceptibility

related

to

diabetes.” in etythrocyte

the

(IDDM)

diabetes

mellitus

to lipoperoxidation,

increased

Lipoperoxidation

incidence

and

non-insulin-

present which

of

was studied

an

in-

might

be

atherosclerosis both

in LDLs

in and

membranes.

_ From the Division of Diubetoloa, Italian National Research Center on Aging, Ancona; the Institute of Biochemisty, Universi~ ofAncona, Ira!v; and the Luborato? of Biochemistry, CHU Rangueil, Toulouse, Ikncr. Submitted May 20. 1993: accepted March 18. 1994. Address reprint requests to Rosa A. Rabini. MO, INRCA Hospital, I iu della Montagnola 164. 60125 Ancona. Ita&. Copyright 1~3I994 by W. B. Saunders Company 00X0495!9414312-0003$03.0010 1470

SUBJECTS AND METHODS We studied 11 IDDM patients (six men and five women aged 38 2 9 years; duration of disease, 13 + 8 years; body mass index, 22.1 2 2.4 kg/m”), 18 NIDDM patients (10 men and eight women, aged 49 2 8 years; duration of disease. 8 ? 5 years: body mass index, 23.2 + 2.7 kg/m?). and 10 sex- and age-matched healthy subjects (body mass index, 22.9 2 2.0 kg/m?). The diagnosis of IDDM and NIDDM was based on the criteria of the World Health Organization.‘? Three IDDM and four NIDDM patients had background retinopathy. No patient was taking lipid-lowering drugs or exhibited microalbuminuria. All subjects selected for the study fulfilled the following inclusion criteria: an absence of hypertension and no smoking habit in the past or at least 1 year of abstention from smoking. Control subjects were recruited from the hospital staff and exhibited normal glucose tolerance as determined by an oral glucose tolerance test. After the selection, both the patients and the control subjects were placed for 2 months on the same normocaloric diet (15% to 20% proteins, 30%’ lipids with a 1:l:l ratio of saturated: monounsaturated:polyunsaturated fat. cholesterol < 300 mgid, 50% to 55% carbohydrates. ascorbic acid 60 to 70 mgid. vitamin E 20 to 30 mgid). The compliance of patients and controls was evaluated by weekly food records. After 2 months of the diet, blood was drawn in the fasting state from the forearm vein for isolation of LDLs and erythrocyte membranes. The following metabolic parameters were evaluated in the same blood samples: plasma glucose, triglycerides (TG). phospholipids (PL), total cholesterol. high-density lipoprotein (HDL) cholesterol, apo A-I and apo B, and hemoglobin A,, (HbAI,). TG, PL, total cholesterol, and HDL cholesterol levels were measured by enzymatic methods. Ii-l’ apo A-I and apo B levels were determined by immunonephelometry.‘h and HbA,, was assayed by high-performance liquid chromatography.” For blood chemistry analyses determined with routine laboratory methods. the maximum intraassay and interassay coefficients of variation were respectively 3.7:; and 4.4%. Results, expressed as the mean + SD. were compared by ANOVA and considered significantly different if P was less than .05.

LDL Isolation LDLs were separated tion according to the

Metabolism,

from total plasma by selective precipitamethod of Alcindor and Antebi” and

Vol43, No 12 (December). 1994: pp 1470.1474